Cell Biology and Translational Medicine
Cell Biology and Translational Medicine
Cell Biology and Translational Medicine
Volume 1376
Series Editor
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Canada
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Preface
In this next volume in the Cell Biology and Translational Medicine series, we
continue to explore the potential utility of stem cells in regenerative medicine.
Among topics explored in this volume are regulatory aspects of stem cells,
differentiation, and organogenesis in both health and disease. One goal of the
series continues to be to highlight timely, often emerging topics and novel
approaches that can accelerate stem cell utility in regenerative medicine.
Certainly, COVID-19-related regulation of stem cell function and potential
stem cell–mediated therapeutic options for COVID are such timely topics
included here.
I remain very grateful to Gonzalo Cordova, the associate editor of the
series, and wish to acknowledge his continued support.
I would also like to acknowledge and thank Mariska van der Stigchel,
Assistant Editor, for her outstanding efforts in helping to bring this volume to
the production stages.
A special thank you goes to Shanthi Ramamoorthy and Rathika Ramkumar
for their outstanding efforts in the production of this volume.
Finally, sincere thanks to the contributors not only for their support of the
series but also for their willingness to share their insights and all their efforts to
capture both the advances and the remaining obstacles in their areas of
research. I trust readers will find their contributions as interesting and helpful
as I have.
v
Contents
vii
viii Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Adv Exp Med Biol - Cell Biology and Translational Medicine (2022) 15: 1–27
https://doi.org/10.1007/5584_2021_675
# Springer Nature Switzerland AG 2021
Published online: 5 November 2021
Molecular Mechanisms
of SARS-CoV-2/COVID-19 Pathogenicity
on the Central Nervous System: Bridging
Experimental Probes to Clinical Evidence
and Therapeutic Interventions
S. A. Groppa, D. Ciolac, D. Gasnaș, and P. Leahu Laboratory of Cerebrovascular Diseases and Epilepsy,
Department of Neurology, Nicolae Testemițanu State Institute of Emergency Medicine, Chisinau, Republic of
University of Medicine and Pharmacy, Chisinau, Republic Moldova
of Moldova
D. Mîrzac
Laboratory of Neurobiology and Medical Genetics, Department of Neurology, Nicolae Testemițanu State
Nicolae Testemițanu State University of Medicine and University of Medicine and Pharmacy, Chisinau, Republic
Pharmacy, Chisinau, Republic of Moldova of Moldova
Department of Neurology, Institute of Emergency Department of Neurology, Institute of Emergency
Medicine, Chisinau, Republic of Moldova Medicine, Chisinau, Republic of Moldova
C. Duarte and C. Garcia A. Movila (*)
Department of Oral Science and Translational Research, Department of Oral Science and Translational Research,
College of Dental Medicine, Nova Southeastern College of Dental Medicine, Nova Southeastern
University, Fort Lauderdale, FL, USA University, Fort Lauderdale, FL, USA
D. Efremova, A. Gasnaș, and T. Bălănuță Institute of Neuro Immune Medicine, Dr. Kiran C. Patel
Department of Neurology, Nicolae Testemițanu State College of Osteopathic Medicine, Nova Southeastern
University of Medicine and Pharmacy, Chisinau, Republic University, Fort Lauderdale, FL, USA
of Moldova e-mail: amovila@nova.edu
Department of Neurology, Institute of Emergency
Medicine, Chisinau, Republic of Moldova
1
2 S. A. Groppa et al.
2020) at the end of 2019 in Hubei Province, development of promising interventions able to
China (Petersen et al. 2020), there has been an counteract the COVID-19-associated CNS injury.
unprecedented global effort to describe the virus In this review, we provide a summarized
and the clinical course of the disease (Burki 2021; update of the pathophysiological mechanisms of
Happi et al. 2021). Identifying the molecular SARS-CoV-2-induced CNS tissue damage,
mechanisms of its pathogenicity has become one with a particular focus on the molecular pathways
of the main goals in understanding and, hope- of neuroinvasion, neuroinflammation, and
fully, controlling the viral spread worldwide. neurodegeneration. We also cover the emerging
SARS-CoV-2, the causative agent of the corona- spectrum of clinical neuroinflammatory and neu-
virus disease 2019 (COVID-19), is a positive- rodegenerative disorders associated to COVID-19
sense single-stranded RNA virus, which belongs and discuss the therapeutic approaches targeting
to the subgenus Sarbecovirus of the genus the molecular pathways aimed to minimize the
β-coronavirus. Its genome is strikingly similar SARS-CoV-2 damage to CNS compartments.
to the bat coronavirus and the receptor-binding
domain of the spike (S) glycoprotein of the
Malayan pangolin coronavirus (Kadam et al. 2 The Neurotropism
2021). of SARS-CoV-2
Although in humans the infection usually
presents itself with systemic and respiratory 2.1 Current Evidence
manifestations, neurological complications have for the Potential Neuroinvasion
been reported as well in up to 40% of COVID-19 Routes of SARS-CoV-2
patients (Jakhmola et al. 2020; Groppa et al. 2020;
Silva et al. 2020). Involvement of the central ner- Several recent studies assessing the CNS
vous system (CNS) was confirmed by the presence alterations in fatal COVID-19 cases have
of SARS-CoV-2 in the cerebrospinal fluid (CSF) provided the first hints into the disease’s
(Moriguchi et al. 2020; Wu et al. 2020a) and brain histopathological tissue morbidities, suggested
parenchyma (Mukerji and Solomon 2021; Serrano new data on the presence of viral RNA in the
et al. 2021), specifically in neuronal and capillary cortical neurons, and revealed the pathological
endothelial cells (Baig et al. 2020). It has been features associated to viral infection with minimal
postulated that the neurovirulence of SARS-CoV- immune cell infiltrate (Puelles et al. 2020;
2 is mediated by its binding to angiotensin- Matschke et al. 2020; Yang and Shen 2020;
converting enzyme 2 (ACE-2) receptors expressed Solomon et al. 2020; Song et al. 2021). However,
by endothelial cells of cerebral capillaries and neu- recent autopsy reports and postmortem brain
ronal and glial cells (Ribeiro et al. 2021). The magnetic resonance imaging (MRI) scans of
SARS-CoV-2 invasion triggers the innate and COVID-19 patients demonstrated mixed results.
adaptive immune responses that drive the Some reports failed to demonstrate the presence
neuroinflammation within the CNS compartments of SARS-CoV-2 and any abnormalities in the
and lead to neuronal cell damage and loss respiratory center of the brain (Coolen et al.
(Wu et al. 2020a; Serrano et al. 2021). 2020; Kantonen et al. 2020), while one autopsy
Neuroinflammation along with accelerated cellular showed massive microglial activation and T cell
senescence and susceptible genetic signatures infiltration in the medulla oblongata (Schurink
might prime the CNS to neurodegeneration and et al. 2020), and recent autopsy studies using
precipitate the occurrence of neurodegenerative electron microscopy found viral particles
diseases (Septyaningtrias and Susilowati 2021; entrapped in dilated vesicles of the neurons in
Duarte et al. 2021). Addressing the key samples from the frontal lobes of COVID-19
mechanisms that modulate the SARS-CoV- patients (Paniz-Mondolfi et al. 2020). Moreover,
2 neuropathogenicity might be translated into the a study using mice overexpressing human ACE-2
identification of therapeutic targets and demonstrated that SARS-CoV-2 neuroinvasion,
4 S. A. Groppa et al.
but not respiratory infection, is actually After SARS-CoV-2 reaches the blood stream,
associated with COVID-19 mortality (Song transcytosis to the BBB is due to the presence of
et al. 2021). Given the multiple reports of CNS the virus in the peripheral circulation paired with
invasion, it is reasonable to discuss first the pos- the slow blood flow within the microvasculature
sible ways by which SARS-CoV-2 can invade that enhances binding of the SARS-CoV-2 (S) pro-
cells of the CNS. tein and the ACE-2 receptors in the capillary
endothelium, thereby promoting the viral trans-
2.1.1 Hematogenic Route port across the basolateral membrane (Baig et al.
The hematogenic route of SARS-CoV- 2020; Dey et al. 2021). Indeed a recent study
2 neuroinvasion involves the circulation of viral reported that viral-like particles were actively
particles in the bloodstream and subsequent pen- budding across the brain endothelial cells,
etration of the blood-brain barrier (BBB) suggesting that the hematogenic route is the
(Desforges et al. 2014; Zubair et al. 2020). The most probable pathway for SARS-CoV-2 entry
latter occurs either through the infection of vas- into the CNS (Paniz-Mondolfi et al. 2020). After
cular endothelial cells and transcytosis or through interacting with ACE-2 in the endothelium of
migration of virus-infected leukocytes into the cerebral blood vessels, SARS-CoV-2 could alter
CNS, also known as the “Trojan horse mecha- the BBB permeability and further facilitate viral
nism” (Fig. 1a) (Zubair et al. 2020; Kim et al. entry into the CNS (Ibrahim Fouad 2021). This
2003). increase in BBB permeability is evidenced by
The virus is likely to enter the blood stream postmortem brain MRI scans of COVID-19
and, subsequently, the CNS through the respira- patients showing brain parenchymal
tory tract via epithelium-to-capillary or abnormalities suggestive of BBB breakdown
pneumocyte-to-capillary connections, which (Coolen et al. 2020) and can be worsened by the
allow the virus into the pulmonary/systemic cir- cytokine storm and systemic inflammatory
culation (Barrantes 2020). SARS-CoV-2 may response with remarkable BBB permeability
also enter the circulation through the gastrointes- effects triggered by SARS-CoV-2 (Li et al.
tinal tract via both endocytic and nonendocytic 2020a; Iadecola et al. 2020a). Furthermore,
mechanisms (Nash and Buchmeier 1997). The SARS-CoV-2 can directly reach the CNS through
gut-brain axis may be key component involved the hematogenic route connecting the nasal
in disorders that affect the CNS (de Mattos mucosa to the anatomically adjacent forebrain
Coelho-Aguiar et al. 2019) since the SARS- (Barrantes 2020).
CoV-2 infects the human intestinal epithelium, The “Trojan horse mechanism” is evidenced
which has a higher expression of ACE-2 by the identification of SARS-CoV-2 in
receptors than the lungs (Lamers et al. 2020; circulating monocytes and lymphocytes from
Xiao et al. 2020). Interestingly, SARS-CoV- infected patients (Gu et al. 2005) and by single-
2 was detected in COVID-19 patients’ rectal cell sequencing where viral RNA was detected in
swabs (Chen et al. 2020a; Tang et al. 2020) and the macrophages from bronchoalveolar lavage
fecal samples (Wu et al. 2020b) and remains fluid of COVID-19 patients, although it is uncer-
detectable in these samples even after negative tain whether the immune cells were infected
results of polymerase chain reaction (PCR) from themselves or had phagocytized virus-infected
the nasopharyngeal swab tests. Moreover, cells (Bost et al. 2020). These infected monocytes
COVID-19 patients with concomitant gastroin- can travel to the blood stream, and the peripheral
testinal symptoms have worse clinical outcomes circulation may bring the virus to other organ
with increased acute respiratory distress and need systems, including the CNS (Gu et al. 2005;
for mechanical ventilation (Jin et al. 2020). Spiegel et al. 2006). Indeed, SARS-CoV-2 can
Molecular Mechanisms of SARS-CoV-2/COVID-19 Pathogenicity on the Central. . . 5
Fig. 1 The SARS-CoV-2-associated mechanisms of basigin (BSG), or neuropilin-1 (NRP1). Through the
neuroinvasion, neuroinflammation, and hematogenic route, SARS-CoV-2 reaches the CNS by
neurodegeneration. (a) Neuroinvasion. The SARS- infecting the cerebral endothelial cells, through
CoV-2 may gain access into the central nervous system compromised blood-brain barrier (BBB) or via circulating
(CNS) via hematogenic and neurogenic routes by binding virus-infected leucocytes, the “Trojan horse” mechanism.
to the angiotensin-converting enzyme 2 (ACE-2) receptor, Through the neurogenic route, SARS-CoV-2 may reach
6 S. A. Groppa et al.
infect dendritic cells, which are known to migrate presence of SARS-CoV-2 RNA and SARS-
to lymph nodes and may be able to disseminate CoV-2 S protein in anatomically distinct regions
the virus (Spiegel et al. 2006). of the nasopharynx and brain by in situ
hybridization and immunohistochemical staining
2.1.2 Neurogenic Route techniques, which suggest that SARS-CoV-
The neurogenic pathway, an important route used 2 could enter the nervous system by crossing the
by neurotropic viruses, involves viral migration to neural-mucosal interface as a result of the close
the CNS by infection of the sensory or motor vicinity of the olfactory mucosal, endothelial, and
nerve endings and consequent use of the retro- nervous tissues, including the olfactory and sen-
grade or anterograde neuronal transport mediated sory nerve endings (Meinhardt et al. 2021). It was
by dynein and kinesin motor proteins to move the also suggested that SARS-CoV-2 might spread
virus toward the neuron body (Swanson 2nd and from the olfactory epithelium to the olfactory
McGavern 2015). The potential neuronal retro- bulb and nerve through endocytosis and exocyto-
grade/anterograde transport and the transsynaptic sis during transsynaptic transfer (Bulfamante
transfer of SARS-CoV-2 are supported by in vitro et al. 2020; Pennisi et al. 2020). Furthermore,
studies that detected the virus within neuronal postmortem brain MRI scans and autopsy of
soma and neurites in a human-induced pluripotent COVID-19 patients also discovered asymmetric
stem cell (iPSC)-derived BrainSphere model and olfactory bulbs, microglia activation, astrogliosis,
in human brain organoids, where SARS-CoV- and T cell infiltration in the olfactory bulb, which
2 exerted metabolic changes through unique indicate that the olfactory neuroepithelium may
pathways compared to other neurotropic viruses, mediate viral entry (Schurink et al. 2020).
such as the Zika virus (Song et al. 2021; Bullen Supporting evidence previously demonstrated
et al. 2020). In addition to the retrograde transport that other coronaviruses are able to enter the
and similar to other coronaviruses, SARS-CoV- brain upon intranasal infection (Netland et al.
2 may exploit the axonal endoplasmic reticulum 2008; Doobay et al. 2007) and that intranasal
of infected neurons to disseminate within the inoculation of SARS-CoV-2 results in a lethal
brain parenchyma (Fenrich et al. 2020). disease with high levels of viral replication in
A clear example of a neurogenic pathway is the brain of mice expressing the human ACE-2
the olfactory neuronal transport (Fig. 1a). receptor (Kumari et al. 2021). Furthermore, a
Recently, Meinhardt et al. demonstrated the more recent study proposed that, in humans,
ä
Fig. 1 (continued) the CNS by infecting the epithelial directed against the neuronal cells and cause cytolysis and
lining of the olfactory mucosa or digestive tract and the demyelination. (c) Neurodegeneration. The migrated
supplying nerve endings with subsequent retrograde neu- immune cells and CNS-resident cells may maintain a
ronal transport toward the CNS. (b) Neuroinflammation. chronic neuroinflammatory milieu within the CNS that
The SARS-CoV-2-triggered innate and adaptive immune causes neuronal damage, demyelination, and axonal loss.
responses may involve the CNS tissue, thereby causing Within the activated microglial cells, the NLR family
neuroinflammation. Pro-inflammatory cytokines (e.g., pyrin domain containing 3 (NLRP3) inflammasome
IL-6, TNF-α) released by peripheral inflammatory cells mediates the release of pro-inflammatory cytokines (e.g.,
(e.g., neutrophils, monocytes, T cells) increase the perme- IL-6, TNF-α), which exert various deleterious effects on
ability of the BBB and activate the CNS-resident cells (i.e., CNS tissue. Activated microglial cells and astrocytes also
microglia and astrocytes). In turn, microglial cells and release reactive oxygen species (ROS) and glutamate,
astrocytes also secrete pro-inflammatory cytokines (e.g., which is responsible for the excitotoxic damage of neuro-
IL-6, IL-8) and chemokines (e.g., CCL-2, CXCL-9), nal cells. Exposure of neurons to SARS-CoV-2 might alter
which alter the BBB integrity and recruit peripheral the function of the endoplasmic reticulum and
leukocytes into the CNS, ultimately resulting in neuronal mitochondria and impair the proteostasis, thereby leading
cell damage and demyelination. Besides antiviral protec- to aggregation of misfolded proteins and neuronal apopto-
tion by destruction of the virus-infected astrocytes and sis, altogether translating into neurodegeneration
microglial cells, the activity of CD8+ T cells might be
Molecular Mechanisms of SARS-CoV-2/COVID-19 Pathogenicity on the Central. . . 7
SARS-CoV-2 CNS entry is due to the infection of and neurons (Paniz-Mondolfi et al. 2020; Lukiw
supporting, non-neuronal cells, which express et al. 2020). Interestingly, ACE-2 is also highly
ACE-2 receptors and the transmembrane serine expressed in the two main regions responsible for
protease 2 (TMPRSS2) (Brann et al. 2020). the regulation of the respiratory cycle, ventrolat-
In addition to the transcribial route and the eral medulla, and nucleus of the solitary tract
olfactory nerve, SARS-CoV-2 might also be (Montalvan et al. 2020). After binding of the S
transferred to the CNS through other peripheral protein to the ACE-2 receptor, subsequent cleav-
nerves such as the vagus nerve, by which lung age by TMPRSS2, cathepsin L, or furin induces
and gut afferents reach the brainstem (Yavarpour- the endocytosis and translocation of SARS-CoV-
Bali and Ghasemi-Kasman 2020; Esposito et al. 2 into the endosomes or direct viral envelope
2020; Li et al. 2020b). The anterograde and retro- fusion with the host cell membrane for cell entry
grade viral transmission from duodenal cells to (Millet 2018; Sanclemente-Alaman et al. 2020).
brainstem neurons has been reported (Parker et al. In fact, it has been reported that the SARS-CoV-
2020), which could be achieved by SARS-CoV- 2 S protein can induce the genetic expression and
2 infection of enterocytes and further transmis- activity of cathepsin L, in vitro (Duarte et al.
sion via the vagus nerve to glial and neuronal 2021).
cells within the enteric nervous system (Esposito One of the main arguments against SARS-
et al. 2020; DosSantos et al. 2020). The trigemi- CoV-2 neuroinvasion is that mRNA levels of
nal nerve, which innervates nociceptive cells in ACE-2 appear to be very low in the CNS (Chen
the nasal cavity and has been successfully tested et al. 2020a; Qi et al. 2020; Sungnak et al. 2020);
for drug transportation (Lochhead et al. 2019), however, widespread expression of ACE-2 was
may also be a route for SARS-CoV-2 migration recently found in both microtubule-associated
to the CNS since it provides sensory nerve protein 2-positive neurons and cells in the neural
endings to the conjunctiva, where SARS-CoV- tube-like structures of the human brain organoids,
2 RNA fragments have been found in a patient indicating that the mRNA levels of ACE-2 do not
with conjunctivitis, and to the taste buds, which accurately reflect the ACE-2 protein expression in
ascend to the trigeminal nuclei and the nuclei of the CNS (Song et al. 2021).
the solitary tract (Chen et al. 2020b). In addition,
the oculomotor and glossopharyngeal nerves are 2.2.2 Dipeptidyl Peptidase-4
believed to serve the same purpose (Keyhan et al. Dipeptidyl peptidase 4 (DPP4), originally known
2020; Wu et al. 2020c). as cluster of differentiation (CD) 26, was
identified as a functional receptor for the Middle
East respiratory syndrome coronavirus, acting as
2.2 CNS Targets of SARS-CoV-2 its main entry pathway (Park et al. 2019). The
DPP4 is generally expressed in human bronchio-
2.2.1 Angiotensin-Converting Enzyme 2 lar and lung epithelial cells (Raj et al. 2013), but it
Numerous studies have established, beyond can also be found in the intravascular portion of
doubt, that the membrane-bound metalloprotease the endothelial cells and in the CSF (Al-Badri
ACE-2 is the SARS-CoV-2 host cell receptor et al. 2018). Furthermore, bioinformatic
(Barrantes 2020). Histochemical and, more approaches combining the prediction of human-
recent, messenger RNA (mRNA) transcriptomic virus protein interactions and protein docking
studies have dissected its cellular localization in based on crystal structures have revealed high
various tissues and proved that SARS-CoV- affinity between the human DPP4 and the S
2 binds to the enzymatic domain of the ACE-2 receptor-binding domain of SARS-CoV-2 (Li et al.
receptor exposed on the surface of several cell 2020b). This observation raised the hypothesis
types, including alveolar cells, intestinal epithe- that SARS-CoV-2 might also use the DPP4
lial cells, endothelial cells, kidney cells, enzyme as a functional receptor to gain entry
monocytes/macrophages, neuroepithelial cells, into the host cells (Vankadari and Wilce 2020).
8 S. A. Groppa et al.
However, another study failed to demonstrate the cell plasma membrane and is essential for SARS-
SARS-CoV-2 binding to DPP-4 or the protective CoV-2 entry to the cellular cytoplasm (Zang et al.
role of DPP4 inhibitors against COVID-19 2020). The olfactory epithelium cells, mainly
(Fadini et al. 2020). sustentacular cells, Bowman’s gland cells, and
basal cells, highly express the TMPRSS2 receptor,
2.2.3 Basigin which is necessary for viral binding, replication,
Basigin (BSG) or CD147, a surface molecule and accumulation; however, TMPRSS2 and
belonging to the immunoglobulin superfamily ACE-2 are, both, absent in the olfactory sensory
that is a potent inducer of matrix metalloproteases neurons (Brann et al. 2020; Bilinska et al. 2020;
(MMPs) and vascular endothelial growth factor, as Fodoulian et al. 2020; Butowt and Bilinska 2020).
well as an important regulator of cell metabolism Therefore, a direct connection between TMPRSS2
(Chen et al. 2010), has been reported to be an and neuroinvasion cannot be established.
alternative entry receptor for SARS-CoV-2 (Ulrich
and Pillat 2020; Wang et al. 2020). It is widely 2.2.5 Neuropilin-1
expressed in epithelial, neuronal, myeloid, and Neuropilin-1 (NRP1), a transmembrane receptor
lymphoid cells (Grass and Toole 2016), which that lacks a cytosolic protein kinase domain, is
might increase the likelihood of infection of multi- also expressed in areas of the CNS, including
ple organs including those in the CNS. The most olfactory-related regions such as the olfactory
robust data supporting the role of BSG as an tubercles and paraolfactory gyri (Davies et al.
alternative receptor for SARS-CoV-2 comes from 2020). The NRP1 receptor has two isoforms: a
a study using co-immunoprecipitation, surface truncated, secreted form and a transmembrane
plasmon resonance, and enzyme-linked immuno- form, which interacts with SARS-CoV-2 and
sorbent assays, which identified BSG as the bind- can serve as an entry factor that potentiates
ing partner of the SARS-CoV-2 S protein that is SARS-CoV-2 infectivity, in vitro (Cantuti-
essential to viral invasion (Wang et al. 2020). Castelvetri et al. 2020; Daly et al. 2020). Its
Notably, this original finding has already been interactions with SARS-CoV-2 and the receptor
translated into an open-label clinical trial of a identification in areas of the CNS suggest the
humanized monoclonal antibody against BSG, potential role of NRP1 as an additional SARS-
meplazumab, which reported striking CoV-2 infection mediator implicated in the neu-
improvements in COVID-19 patients (Bian et al. rological manifestations of COVID-19. While
2020). However, a recent report was unable to find NRP1 alone promotes SARS-CoV-2 entry and
evidence supporting the role of BSG as a putative infection, its co-expression with ACE-2 and
binding receptor for the S protein or evidence of TMPRSS2 markedly potentiates this process
direct interaction between the S protein and either (Cantuti-Castelvetri et al. 2020; Daly et al.
of the two common BSG isoforms (Shilts et al. 2020). Moreover, autopsy studies in COVID-19
2021). A recent genomic study investigating gene patients, as well as mice studies, showed that
variants linked to SARS-CoV-2 infection failed to SARS-CoV-2-infected NRP1-positive cells in
find evidence of BSG variant enrichment in the olfactory epithelium, tract, and bulb can medi-
COVID-19 patients despite identifying ate the transport of virus-sized particles from the
associations to more established viral entry factors, intranasal cavity to the brain (Cantuti-Castelvetri
such as the TMPRSS2 (Latini et al. 2020). et al. 2020).
3A), as well as increase the activation of MMPs microglia and MHC class I antigens in oligoden-
and cyclooxygenase-2 (Costela-Ruiz et al. 2020). drocytes (Septyaningtrias and Susilowati 2021).
Furthermore, IL-17 can induce neurovascular Hence, the CD8+ T cells mediate viral clearance
endothelial expression of MCP1 and C-X-C from resident glial cells and CD4+ T cells from
motif chemokine ligand (CXCL) 1, which facili- oligodendrocytes (Savarin and Bergmann 2018).
tate the traffic of activated Th17 cells into the Notwithstanding their antiviral activity, T cell
CNS (Wojkowska et al. 2017). Additionally, responses are also associated with CNS tissue
IL-17 can cause neuronal toxicity and injury by injury by inducing cytolysis and demyelination
activation of the nuclear factor kappa-light chain and instructing myeloid cells to initiate tissue
enhancer of activated B cells (NF-kB) (Najjar damage (Savarin and Bergmann 2018). Com-
et al. 2020). pared to non-hospitalized COVID-19 patients,
the hospitalized ones displayed increased blood
3.1.2 Blood-Brain Barrier Disruption levels of cytotoxic follicular helper cells and cyto-
Several mechanisms have been identified, which toxic T helper cells and reduced levels of regu-
might compromise the integrity of the BBB and latory T cells (Meckiff et al. 2020). Moreover,
promote inflammation within the CNS infiltration of T cells in perivascular spaces and
compartments during the onset of SARS-CoV- brain parenchyma was detected in patients with
2 infection. First, the main mediators of the cyto- SARS-CoV-2 infection (Bryce et al. 2020;
kine storm, IL-6, TNFα, and IL-17, were shown Hanley et al. 2020). However, the relevance of
to alter the permeability of the BBB under exper- this identified T cell profile in patients with
imental conditions (Najjar et al. 2020). Second, neuroinflammatory complications remains to be
similar to the infection of endothelial cells and elucidated.
inflammation within the peripheral vessels,
SARS-CoV-2 might induce the inflammation of 3.1.4 Autoimmunity
the cerebral endothelial cells and, thereby, disrupt Besides the cytokine- and adaptive cell-mediated
the BBB (Teuwen et al. 2020). Third, the barrier inflammatory injury to the CNS tissues, the
functions of the BBB might be altered by SARS-CoV-2 infection may as well induce auto-
activated microglial cells, which can disintegrate immune responses. Currently, the underlying
the tight junctions of the BBB endothelium by mechanisms of SARS-CoV-2-triggered autoim-
releasing pro-inflammatory cytokines and induc- mune CNS complications are poorly understood;
ing MMP activity (Bongetta et al. 2020). Follow- however, the cross-reactivity between the SARS-
ing BBB disruption, the interaction between the CoV-2 and CNS epitopes and “bystander activa-
peripheral immune mediators and the tion” of pre-primed auto-reactive T cells might be
CNS-resident cells translates into impaired neu- postulated (Ehrenfeld et al. 2020). In line with
rotransmission, glutamate-mediated neurotoxic- molecular mimicry hypothesis, immune
ity, synaptic alterations, and neuroglial cell responses triggered by SARS-CoV-2 infection
death (Septyaningtrias and Susilowati 2021). and directed to combat the virus might cross-
react with the self-antigens in the cerebrovascular
3.1.3 Adaptive Immunity Responses endothelial cells or neurons and result in an auto-
The cytotoxic CD8+ T cells are essential players immune inflammation. The inflamed CNS tissue
in antiviral immune protection through IFN-γ, might generate damage-associated molecular
granzyme B, and perforin, which destroy the patterns (DAMPs) and autoantigens that stimulate
virus-infected astrocytes and microglial cells the inflammasome platforms and predispose to
(Savarin and Bergmann 2018). Meanwhile, autoimmunity (Talotta and Robertson 2020).
CD4+ T cells boost the cytolytic activity of The S protein was shown to play a distinct role
CD8+ T cells within the CNS by secreting in induction of immune-mediated neuropathology
IFN-γ and increasing the expression of major given its high immunogenic potential (Lyons-
histocompatibility complex (MHC) class II in Weiler 2020). Autoimmune complications of the
Molecular Mechanisms of SARS-CoV-2/COVID-19 Pathogenicity on the Central. . . 11
CNS so far reported are: anti-N-methyl-D-aspar- astrocytes in vitro through a mechanism that
tate receptor encephalitis (Monti et al. 2020; involves a spike-NRP1 interaction (Tavcar et al.
Panariello et al. 2020), Bickerstaff encephalitis 2021). Astrocytes play important roles in
(Llorente Ayuso et al. 2021; Ayuso et al. 2020), orchestrating the immune responses to neuro-
autoimmune meningoencephalitis, and acute infection and neuroinflammation since reactive
disseminated encephalomyelitis (ADEM) astrocytes promote immune-mediated inflamma-
(Paterson et al. 2020). tion and neuronal damage by secretion of
pro-inflammatory cytokines (Tremblay et al.
2020). Astrocytes produce IL-1β, IL-6, IL-8,
3.2 Compartmentalized Neuroglial IL-12, IL-17, and TNF-α that exert neurotoxic
Responses effects and alter neuronal survival (Choi et al.
2014; Sofroniew 2014). Their detrimental effects
The CNS-resident cells may be involved in are enhanced by the release of other cytotoxic
neuroinflammation either by circulating cytokines factors such as nitric oxide (NO), reactive oxygen
or viral proteins leaked into the CNS through the species (ROS), nitrogen oxygen species (NOS),
breached BBB (Fig. 1b). In the first scenario, and glutamate (Tremblay et al. 2020). Further-
pro-inflammatory cytokines from the systemic cir- more, damaged neurons release the adenosine
culation stimulate the microglial cells and triphosphate, which activates the purinergic
astrocytes to produce cytokines (Wu et al. receptor P2X7 (P2X7) expressed by microglia
2020a). In the second scenario, viral proteins and and astrocytes and results in increased Ca2+ influx
molecular complexes (e.g., nuclear protein high and glutamate release (Ribeiro et al. 2021). This
mobility group box 1) from peripheral tissue glutamate level increase is the main mechanism
injured cells might reach the CNS and behave as of excitotoxicity that leads to neuronal cell dam-
DAMPs and pathogen-associated molecular age and subsequent neurodegeneration
patterns (PAMPs) (Iadecola et al. 2020a). Conse- (Mahmoud et al. 2019). In addition, the
quently, both DAMPs and PAMPs may launch an astrocyte-released chemokines CXCL10,
innate immune response in the cells expressing CXCL12, chemokine (C-C motif) ligand (CCL)
toll-like receptors (TLRs), such as CNS-resident 2, and CCL5 recruit peripheral leukocytes and
macrophages, microglia, and astrocytes, which resident microglia into the inflamed CNS (Choi
release pro-inflammatory cytokines (Iadecola et al. 2014; Sofroniew 2014). Increased levels of
et al. 2020b). Both scenarios result in a CCL2 mRNA were identified in astrocytes fol-
neuroinflammatory environment (Dogan et al. lowing human coronavirus OC43 (HCoV-OC43)
2020), ultimately leading to CNS tissue damage. infection (Aghagoli et al. 2020), while binding of
MCP1 to the C-C chemokine receptor type 2 is
3.2.1 Astrocyte Responses known to alter the permeability of the BBB
Despite the presence of ACE-2 receptors in (Stamatovic et al. 2005). Postmortem studies in
astrocytes (Chen et al. 2021), it is unclear whether deceased COVID-19 patients are indicative of
SARS-CoV-2 can infect the astrocytes. Prelimi- reactive astrogliosis due to increased expression
nary data from a recent histopathological study of glial fibrillary acidic protein in the white matter
found that the affected brain tissue displayed foci (Reichard et al. 2020). Aside from the many
of SARS-CoV-2 infection and replication, partic- inflammatory and neurodegenerative roles men-
ularly in astrocytes (Tavcar et al. 2021). The tioned above, astrocytes have a protective role by
susceptibility of human astrocytes to SARS- secretion of anti-inflammatory cytokines, such as
CoV-2 infection was confirmed in the same IL-4, IL-5, and TGF-β, and neurotrophic factors,
study, showing that the replication of SARS- such as the brain-derived neurotrophic factor
CoV-2 occurs in neural stem cell-derived human (Sofroniew 2014).
12 S. A. Groppa et al.
characterized by an abnormal signal over multiple with SARS-CoV-2 infection has been reported in
segments of the spinal cord (Garg et al. 2021). several case studies (Paterson et al. 2020; Parsons
Additionally, patients presented a nonspecific et al. 2020; Novi et al. 2020). The clinical
CSF inflammatory profile with elevated protein manifestations ranged from headache, sensory
levels and lymphocytic pleocytosis, while SARS- and motor deficits to cranial nerve involvement,
CoV-2 RNA was detectable in the CSF of a minor- encephalopathy, and seizures, while the MRI
ity of patients (Garg et al. 2021). Patients received showed hyperintense lesions, with some of the
corticosteroids, IVIG, or plasma exchange with patients developing hemorrhagic changes and a
favorable outcomes in majority of patients. few presenting concomitant myelitis (Paterson
et al. 2020). No traces of SARS-CoV-2 RNA
3.3.4 Acute Necrotizing Myelitis (ANM) were detected in the CSF of patients or in the
Acute necrotizing myelitis is a rare inflammatory neuropathological samples of the brain tissue
disorder of the spinal cord characterized by (Paterson et al. 2020). The applied treatment
hemorrhages and cavitation on spinal MRI. included steroids or IVIG with outcomes varying
Along with ATM, two cases of ANM associated from death to partial recovery (Paterson et al.
with SARS-CoV-2 infection were recently 2020; Reichard et al. 2020; Parsons et al. 2020).
described (Sotoca and Rodríguez-Álvarez 2020;
Maideniuc and Memon 2020). The clinical pre-
sentation was marked by limb weakness and sen- 3.4 Illustrative Case 1
sory and bladder symptoms, while the MRI
findings for both cases presented as LETM, with A young male in his 40s complaining of fever,
T2-hyperintense lesions, spinal cord swelling, breathlessness, and cough for the last 2 days was
and areas of necrosis, and the CSF showed high admitted to the COVID-19 intensive care unit
protein levels and cell counts and no oligoclonal (ICU) with altered mental status, bulbar palsy,
bands (Sotoca and Rodríguez-Álvarez 2020; and bilateral limb ataxia. The naso /oropharyn-
Maideniuc and Memon 2020). Both patients geal swabs were positive for SARS-CoV-2 infec-
responded positively to high-dose steroids and tion, and the CSF analysis revealed lymphocytic
plasma exchange. pleocytosis and presence of red blood cells, with-
out detectable SARS-CoV-2 RNA. His chest
3.3.5 Acute Disseminated computed tomography (CT) showed bilateral
Encephalomyelitis (ADEM) pneumonia with ground glass appearance, and
The ADEM is a demyelinating, usually the brain MRI was characterized by widespread
monophasic, disorder occurring after a viral cerebellar and thalamic lesions (Fig. 2). Other
infection or vaccination and manifesting clini- potential causes, including herpes simplex virus
cally with multifocal deficits and encephalopathy 1 and 2, cytomegalovirus, and Epstein-Barr virus,
(Pohl et al. 2016). Neuroimaging presentation were excluded. The clinical, CSF and imaging
includes bilateral and asymmetric lesions within findings were indicative of an ANE. During hos-
the subcortical white matter, cortical gray-white pitalization, along with cerebellar and brainstem
matter junction, thalamus, basal ganglia, cerebel- symptomatology, cognitive and behavioral
lum, and brainstem (Pohl et al. 2016). Spinal cord impairment dominated the clinical presentation.
lesions occur in one third of the patients and The patient showed good response upon pulse
involve multiple segments (Pohl et al. 2016). steroid and sedation therapy, with a favorable
The classical presentation of ADEM in patients outcome at discharge.
Molecular Mechanisms of SARS-CoV-2/COVID-19 Pathogenicity on the Central. . . 15
Fig. 2 Brain imaging from the illustrative case (a, e), restricted diffusion (cytotoxic edema) on diffusion-
1 (acute necrotizing encephalopathy). Bilateral cerebel- weighted images (b, f) and apparent diffusion coefficient
lar and thalamic lesions (arrows) displaying a hyperintense maps (c, g), and hemorrhages on susceptibility-weighted
signal on axial fluid-attenuated inversion recovery images images (d, h); personal courtesy of DC
examination of patients with SARS-CoV-2 infec- Similarly, higher levels of glial fibrillary acidic
tion (Hanley et al. 2020). Persistent microglial protein, a marker of astrocyte injury, were
activation is associated with neurotoxicity, cellu- detected in moderate and severe SARS-CoV-
lar senescence, and subsequent progression of 2 infection (Kanberg et al. 2020). This data aligns
neurodegenerative disorders (Hickman et al. with a previous report of axonal damage mediated
2018). Damage by microglia during SARS- by CD4+ and CD8+ T cells that has been
CoV-2 infection could be mediated by activated observed in areas of demyelination and in adja-
microglial NLRP3 inflammasome, which cent areas with intact myelin after coronavirus
promotes the production of pro-inflammatory infection (Dandekar et al. 2001).
cytokines and the aggregation of pathogenic Considering all the aforementioned, it can be
peptides, as well as mitochondrial dysfunction postulated that the intricate interplay between the
and apoptosis, which contribute to virus-induced peripheral and central immune
neurodegeneration (Ising et al. 2019). responses mediated by the CNS-resident and
The contribution of T and B cells from the adaptive immune cells and their cytokines
adaptive immune system to the neurodegenera- converges on common neuroinflammatory
tive processes following the SARS-CoV-2 infec- pathways that drive the pathogenesis of
tion is less clear; however, perivascular and neurodegeneration.
pericapillary infiltration of T cells without B
cells in severe cases of COVID-19 was evidenced 4.1.2 Accelerated Neurosenescence
(Hanley et al. 2020). T cell infiltration is fre- Cellular senescence can contribute to neurode-
quently observed in neurodegenerative diseases generative processes through several
such as Parkinson’s disease (PD) and mechanisms, including chronic inflammation,
Alzheimer’s disease (AD) (Hong et al. 2016), promotion of oxidative stress and mitochondrial
and similar autoimmune mechanisms of dysfunction, reduction of the CNS regenerative
neurodegeneration related to coronavirus infec- potential, and loss of neuronal function
tion were previously suggested by the identifica- (Martínez-Cué and Rueda 2020; López-Otín
tion of anti-CoV antibodies in the CSF of PD et al. 2013). Viral infections, including the
patients (Fazzini et al. 1992). Therefore, it is SARS-CoV infection, interfere with many of the
reasonable to hypothesize that SARS-CoV-2- pathways involved in cellular aging (López-Otín
induced autoimmune responses within the CNS et al. 2013). Some of those pathways involve
tissue might promote the neurodegenerative pro- ACE-2, DPP4, and furin, the receptors used by
cesses as well. SARS-CoV-2 to enter the host cells, which were
Neutrophils contributed to oligodendrocyte previously found to be implicated in the accelera-
loss and demyelination through a CXCL1- tion of cellular senescence (Sfera et al. 2020). The
dependent mechanism, in mouse models of viral SARS-CoV-2 binding to ACE-2 is likely to
exposure (Marro et al. 2016). Such apoptosis of impair the hydrolysis of angiotensin II and further
oligodendrocytes can occur upon the activation of promote its deleterious effects on cellular physi-
mitochondrial pathways with caspase-8 as ology via oxidative stress and apoptosis, which in
upstream signal (Liu et al. 2006). Since ACE-2 turn affects neurogenesis, neuronal differentia-
is expressed by oligodendrocyte progenitor cells tion, and neuronal survival (Sfera et al. 2020).
(Chen et al. 2021), SARS-CoV-2 could poten- Another ACE-2-mediated mechanism leading to
tially affect the differentiation of oligoden- neurodegeneration is chronic inflammation
drocytes and aggravate the demyelination. through the release of ROS and
Indeed, in patients with severe SARS-CoV- pro-inflammatory mediators from the activated
2 infection, the levels of neurofilament light glial cells (Abiodun and Ola 2020). Like
chain, a marker of axonal injury and axonal ACE-2, DPP4 is also engaged in cellular aging
neurodegeneration, were found to be high and processes. It is expressed on the surface of
increased over time (Kanberg et al. 2020). senescent cells and promotes their elimination
Molecular Mechanisms of SARS-CoV-2/COVID-19 Pathogenicity on the Central. . . 17
probable PD after SARS-CoV-2 infection was profile of CJD, including impaired comprehension,
described in a young male, who developed par- myoclonic jerks, and mutism (Young et al. 2020).
kinsonian motor symptoms, including The second case features an elderly patient with a
bradykinesia, tremor, and rigidity, shortly after typical neuropsychiatric presentation of sporadic
the acute onset of respiratory manifestations CJD (myoclonus, cognitive decline, hallucinations,
(Cohen et al. 2020). and delusions), which clinically deteriorated during
the hospitalization and died soon thereafter
Alzheimer’s Disease Dementia was found to be (McMurran et al. 2020). The SARS-CoV-2-
a risk factor for higher mortality rates related to induced inflammatory (IL-1, IL-6, IL-12, INF-γ,
the SARS-CoV-2 infection (Bianchetti et al. and TNF-α) and immune (activated microglia)
2020). Increased levels of pro-inflammatory responses were suggested to precipitate and/or
cytokines, including IL-1 and IL-6, in the elderly accelerate the prion-driven neurodegeneration
(Rea et al. 2018) may account for exaggerated (Young et al. 2020; Mabbott et al. 2020). High
immune responses that might increase the proba- levels of TNF-α and INF-γ, the cytokines found
bility of poor outcomes following the SARS- to correlate with viral loads in SARS-CoV-2 infec-
CoV-2 infection. Worsening of neuropsychiatric tion, stimulate reactive astrocytes with neurotoxic
symptoms, mainly agitation, apathy, and aberrant effects, thereby accelerating the neurodegenerative
motor activity, was observed in AD patients with processes in prion diseases (Mabbott et al. 2020).
concomitant SARS-CoV-2 infection (Lara et al.
2020). As patients recovering from the SARS- Future experimental and clinical studies will
CoV-2 infection display a range of cognitive reveal the underlying molecular mechanisms and
symptoms (Hellmuth et al. 2021), its long-term the long-term implications of SARS-CoV-2 in
consequences on cognitive performance might as neurodegeneration.
well be expected. A synergistic effect of systemic
inflammation and brain amyloid-stimulated IFN
response was proposed to exacerbate the neuro- 4.3 Illustrative Case 2
degenerative processes and clinical symptomatol-
ogy in AD patients (Naughton et al. 2020; Ferini- A female patient in her 60s presenting with head-
Strambi and Salsone 2020). As a part of the CNS ache, fever, dry cough, and shortness of breath
innate immune system, amyloid is known to entrap was admitted to the COVID-19 ICU. Her nasal
viral particles and consequently induce microglial and oropharyngeal swabs were positive for
activation and type I IFN responses (Naughton SARS-CoV-2 infection, and her chest CT
et al. 2020). A subset of microglial cells, the “neu- revealed bilateral infiltrative consolidations.
rodegenerative phenotype,” associated with the Blood analyses were unremarkable, except for
amyloid plaques is characterized by high expres- high levels of C-reactive protein. Two months
sion levels of IFN-related genes, which further prior to the admission, she was diagnosed with
promote the pro-inflammatory responses and syn- sporadic CJD manifesting with ataxia, myoclo-
apse degradation (Naughton et al. 2020). nus, and cognitive impairment, yet she was able
to walk and carry out some of the daily activities.
Creutzfeldt-Jakob Disease (CJD) Systematic The brain diffusion-weighted MRI showed exten-
studies on the possible effects of SARS-CoV- sive signal abnormality over the cerebral cortex
2 infection on the evolution of CJD are currently and basal ganglia bilaterally (Fig. 3a–d). After her
lacking, yet preliminary insights were provided by respiratory symptoms improved, she was trans-
several case reports (Young et al. 2020; McMurran ferred to the general neurology ward. However,
et al. 2020). The first case described a patient with during the hospitalization her neurological status
the concurrent onset of SARS-CoV-2 infection and significantly deteriorated – she was unable to
CJD, which initially presented with fever and con- walk, her cognitive and behavioral manifestations
fusion and, later, developed a full-blown clinical became worse, and epileptic seizures emerged.
Molecular Mechanisms of SARS-CoV-2/COVID-19 Pathogenicity on the Central. . . 19
Fig. 3 Brain imaging from illustrative case thalamus pulvinar before the SARS-CoV-2 infection (a–
2 (Creutzfeldt-Jakob disease). Diffusion-weighted d). Repeated MRI performed 1 month after the SARS-
images displaying a hyperintense signal in the cortical CoV-2 infection was marked by a more enhanced signal
ribbon over the frontal, parietal, insular, and cingulate (arrows) over the same regions (e–h); personal courtesy
cortices, as well as bilateral putamina, caudate, and of DC
On repeated MRI, a more intense signal over the responses (Riva et al. 2020; Vabret et al. 2020).
cortical and subcortical structures, as compared to Pharmacological therapies, commonly used for
the previous MRI scan, was observed (Fig. 3e–h). neurological complications in other viral
Following a regular seizure, she had respiratory infections, have also been applied in the treatment
depression that required transfer to the ICU and of SARS-CoV-2-associated neuroinflammatory
intubation. Unfortunately, after several days of manifestations, including steroids, IVIG, and
mechanical ventilation, she passed away despite plasmapheresis for ANE, ATM, ANM, ADEM,
the efforts to resuscitate her. and autoimmune encephalitis (Keyhanian et al.
2020). However, several other medications
aimed to modulate the immune responses to
5 Therapeutic Approaches SARS-CoV-2 infection and induce viral clear-
Counteracting Neuronal ance, like remdesivir, hydroxychloroquine,
Damage lopinavir/ritonavir, tocilizumab, interferons, and
convalescent plasma, have shown some beneficial
Several major therapeutic approaches might effects on disease activity (Chibber et al. 2020)
reduce the harmful effects of SARS-CoV-2 infec- and might be used for preventing/treatment of
tion on the CNS via direct or indirect neuroinflammatory conditions.
mechanisms. Vaccination is used to prevent the Remdesivir received the FDA authorization as
infection and/or reduce its severity, while an emergency medication for severely ill
antiviral therapy interferes with the cell’s life hospitalized adult and pediatric patients with con-
cycle, and treatment that targets the immune firmed or suspected SARS-CoV-2 infection
20 S. A. Groppa et al.
the absorption of non-heme iron in the duode- TMPRSS4, and NRP1. The mechanisms of
num, and, by suppressing its gene expression, SARS-CoV-2 neuropathogenicity range from
ARBs might offer a protective effect against the systemic hyperinflammation, activation of innate
SARS-CoV-2 infection at this level (Sfera et al. and adaptive immune signaling pathways, and
2020). Consequently, ARBs could be promising triggering para-infectious autoimmunity to direct
therapeutics for CNS complications and are being viral damage of the CNS tissue, accelerated
tested in ongoing clinical trials in COVID-19 neurosenescence, and neurodegeneration. Several
patients (Sfera et al. 2020). Since ARBs were therapeutic approaches selectively targeting these
demonstrated to also enhance the activity of neuropathogenic mechanisms have been pro-
p53, they might decrease the development of posed and currently are being explored for poten-
cellular and immune senescence, thereby reduc- tial clinical applications. Future studies will be
ing the risk of neurodegeneration (Sfera et al. essential in elucidating the molecular machinery
2020). involved in the SARS-CoV-2-induced CNS
injury and will open new avenues to combat its
Gliptins or DPP4 inhibitors used to prolong the short- and long-term consequences in the post-
incretin half-life, potentiate meal-induced insulin COVID-19 era.
secretion, and treat type 2 diabetes (Drucker 2006)
have shown a variety of effects that could be benefi- Acknowledgements This work was supported by a Nova
cial to COVID-19 pathology in rodent models Southeastern University President Faculty Research
Development Grant and NIH Grants R01AG-064003,
(Mehta et al. 2020). At least three parallel-group
K02AG-068595, R15DE-027153, and R03DE-028699
randomized controlled trials investigating the effects (A.M.).
of DPP4 inhibitors on the prognosis for COVID-19
are currently ongoing. Two of them are examining
the effects of linagliptin added to background insulin
therapy, while the third one is the open-label “Effect References
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https://doi.org/10.1007/5584_2021_665
# Springer Nature Switzerland AG 2021
Published online: 16 December 2021
29
30 H. Raji et al.
4.40, 0.77, P ¼ 0.0052, I2 ¼ 71%) in the illnesses such as Middle East respiratory syn-
lung using the Evans blue extravasation tech- drome (MERS) and severe acute respiratory syn-
nique significantly compared with the model drome (SARS) (Chathappady House et al. 2021).
controls. This systematic review and meta- A recent coronavirus of severe acute respiratory
analysis revealed that the PBM therapy does syndrome coronavirus 2 (SARS-CoV-2), a new
utilize beneficial anti-inflammatory effect, mod- type of this family that was introduced to the
ulation of the immune system, lung permeabil- world in late 2019, is the cause of COVID-19
ity, or bronchoalveolar lavage on lung damage disease (Lai et al. 2020). The body’s response to
in both animal models and clinical studies. How- COVID-19 disease depends on a number of
ever, animal model and clinical studies appear factors, including age, sex, immune system,
limited considering the quality of the included HLA profile, number of ACE2 receptors, envi-
evidences; therefore, large clinical trials are still ronmental factors, underlying diseases, and other
required. unknown factors (Schultze and Aschenbrenner
2021; Haybar et al. 2020). Preliminary studies
also show that many of the fatalities of COVID-
Keywords
19 are due to over-activity of the immune system,
Anti-inflammatory · COVID-19 · Meta- which is known as the cytokine storm (Ragab
analysis · SARS-CoV-2 et al. 2020).
The lethal response of the immune system to the
SARS-CoV-2 is a major issue that has been
discussed since the beginning of the disease
Abbreviations pandemics and can be the cause of many COVID-
19-related deaths (Costela-Ruiz et al. 2020). The
ARDS Acute respiratory distress syndrome
risk of COVID-19-related acute respiratory distress
BALF Bronchoalveolar lavage fluid
syndrome (ARDS) in patients during hospitaliza-
LLLT Low-level laser therapy
tion was directly related to increased neutrophil
MERS Middle East respiratory syndrome
count and decreased lymphocyte count (Gibson
MOOSE Meta-analyses of Observational
et al. 2020). Measurements of interleukin levels
Studies in Epidemiology
indicate the extent of the cytokine storm in
MPO Myeloperoxidase
COVID-19 and are associated with disease severity
PBM Photobiomodulation
(Leisman et al. 2020). Large amounts of cytokines
PML Pulmonary microvascular leakage
can cause widespread (systemic) inflammation that
PRISMA Preferred Reporting Items for
can damage various organs in the body and lead to
Systematic Reviews and Meta-
multi-organ dysfunction syndrome (Rowaiye et al.
analyses
2021).
SARS Severe acute respiratory syndrome
Currently, there is no treatment protocol as the
SARS- Severe acute respiratory syndrome
gold standard for COVID-19 disease, and only
CoV-2 coronavirus 2
supportive protocols that can reduce the inflam-
SD Standard deviation
mation in the severe conditions are often
SMD Standardized mean difference
recommended (Ramphul et al. 2021). There is
WOS ISI web of science
substantial debate about the beneficial effects of
antiviral drugs, while dexamethasone has recently
been shown to be effective in reducing mortality
1 Introduction (Ranjbar et al. 2021).
COVID-19 disease causes ARDS by inducing
Coronaviruses are a large family of viruses that extensive damage to lung tissue; so far, histologi-
cause a range of respiratory infections in humans, cal results obtained from lung biopsy of patients
ranging from the common cold to more severe with COVID-19 showed extensive alveolar
The Probable Protective Effect of Photobiomodulation on the Inflammation of. . . 31
microvascular leakage (PML) were included. cells. Two authors (FR and HR) independently
Studies with no induced lung injury and cytokine performed the title and abstract screening. Any
measurement not in lung tissue, not using PBM disagreement was resolved either by double
therapy, were excluded. We collected information checking the reference paper or discussion with
regarding all outcome measures. First outcome of a third author. Two authors (FR and HR) inde-
interest includes assessment of IL-1β, TNF-α, pendently conducted the methodological quality
IL-10, IL-6, MPO activity, and PML from lung assessment with especial consideration to poten-
tissue. Second outcomes were number of cells in tial sources of risk of bias. We used the Cochrane
bronchoalveolar lavage fluid (BALF), including Collaboration’s quality assessment tool for risk of
neutrophils, macrophages, lymphocytes, and total bias assessment in RCTs (Higgins et al. 2011).
The Probable Protective Effect of Photobiomodulation on the Inflammation of. . . 33
Of the 438 studies found through initial searches, IL-6 Of the 13 included studies, 3 measured IL-6
13 met the inclusion criteria (Fig. 1) (Aimbire (da Cunha Moraes et al. 2018, Miranda da Silva
et al. 2005, 2006; Aimbire et al. 2008; da Cunha et al. 2015, Oliveira Jr. et al. 2014), which were
Moraes et al. 2018; de Lima et al. 2010, de Lima included in the meta-analysis. PBM therapy
et al. 2011a, b, 2013a, b, de Lima et al. 2014; reduced IL-6 significantly compared with the
Fazza et al. 2020; Miranda da Silva et al. 2015; model controls (SMD:-4.20, 95% CI: 6.42,
Oliveira Jr. et al. 2014). After applying the exclu- 1.97, P ¼ 0.0002, I2 ¼ 88%) (Fig. 2).
sion criteria, the main properties of 13 articles on
384 animals were included in this meta-analysis IL-10 Of the 13 included studies, 4 measured
with a wide range of species including rat IL-10 (da Cunha Moraes et al. 2018, de Lima,
(n ¼ 10) and rabbit (n ¼ 3) (Table 2). Moreira, et al. 2011, Miranda da Silva et al. 2015,
34 H. Raji et al.
Duplicated records
Records af ter duplicates removed excluded
(n = 426) (n =12)
Screening
Records excluded
Full-text articles
Full-text articles assessed excluded, with reasons
Eligibility
Letter = 65
Oliveira Jr. et al. 2014), which were included in significantly compared with the model controls
the meta-analysis. PBM therapy increased IL-10 (SMD:-2.13, 95% CI: 3.38, 0.87,
significantly compared with the model controls P ¼ 0.0009, I2 ¼ 64%) (Fig. 3a).
(SMD:-4.65, 95% CI: 6.15, 3.16,
P < 0.00001, I2 ¼ 62%) (Fig. 2). Pulmonary Microvascular Leakage (PML) Of
the 13 included studies, 3 measured
Myeloperoxidase (MPO) Activity Of the (4 measurements) (Aimbire et al. 2008, de Lima
13 included studies, 4 measured MPO activity in et al. 2013a, Miranda da Silva et al. 2015) PML in
lung tissue that was included in the meta-analysis lung tissue that were included in the meta-
(Aimbire et al. 2008, de Lima et al. 2014, analysis. PBM therapy reduced vascular perme-
Miranda da Silva et al. 2015, de Lima, Vitoretti, ability in the lung using the Evans blue extrava-
et al. 2013). PBM therapy reduced MPO activity sation technique significantly compared with the
The Probable Protective Effect of Photobiomodulation on the Inflammation of. . . 35
Fig. 2 Comparison of the effect on TNF-α, IL-1β, IL-6, necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-
and IL-10 between PBM therapy and control in the animal 1β; IL-10, interleukin-10; SMD, standardized mean differ-
lung injury models. The figure represents the SMD result ence; CI, confidence interval; PBM, photobiomodulation;
of the overall experimental data, and the horizontal lines SD, standard deviation; IV, independent variable
represent the 95% CIs for each study. TNF-α, tumor
model controls (SMD:-2.59, 95% CI: 4.40, et al. 2020, Oliveira Jr. et al. 2014). The analysis
0.77, P ¼ 0.0052, I2 ¼ 71%) (Fig. 3b). revealed that PBM therapy reduced the number of
neutrophils significantly compared with the model
Neutrophils Of the 13 included studies, controls (SMD:-3.71, 95% CI: 5.36, 2.05,
7 measured the number of neutrophils in BALF P < 0.00001, I2 ¼ 77%) (Fig. 4).
(Aimbire et al. 2008, da Cunha Moraes et al. 2018,
de Lima et al. 2011a, de Lima et al. 2011b, Fazza Macrophages Of the 13 included studies,
et al. 2020, Miranda da Silva et al. 2015, Oliveira 3 measured the number of macrophages in
Jr. et al. 2014), which were included in the meta- BALF (da Cunha Moraes et al. 2018, Fazza
analysis. Of this seven studies, two studies include et al. 2020, Miranda da Silva et al. 2015), which
two different groups of PBM and controls (Fazza were included in the meta-analysis. The analysis
The Probable Protective Effect of Photobiomodulation on the Inflammation of. . . 37
Fig. 3 Comparison of the effect on the MPO activity in experimental data, and the horizontal lines represent the
lung tissue (a) and vascular permeability in the lung using 95% CIs for each study. SMD standardized mean differ-
the Evans blue extravasation technique (b) between PBM ence, CI confidence interval, PBM photobiomodulation,
therapy and control in the animal lung injury models. The SD standard deviation, IV independent variable
figure represents the SMD result of the overall
revealed that PBM therapy reduced the number of 3.3 Sensitivity Analyses
macrophages significantly compared with the
model controls (SMD:-1.43, 95% CI: 2.83, There was a substantial variation in the
0.02, P ¼ 0.05, I2 ¼ 86%) (Fig. 4). comparators of included studies; thus, we
performed a sensitivity analysis of the effects of
Lymphocytes Of the 13 included studies, various comparators on study outcomes. We
5 measured the number of lymphocytes in found no statistically significant differences
BALF (da Cunha Moraes et al. 2018, de Lima, between PBM therapy and control in sensitivity
Albertini, et al. 2013, Fazza et al. 2020, Miranda analysis. We observed any publication bias using
da Silva et al. 2015, Oliveira Jr. et al. 2014), funnel plots for immunologic factor measures and
which were included in the meta-analysis. The BALF cell outcomes, in which all plots appeared
analysis revealed that PBM therapy reduced the to be non-symmetrical with obvious publication
number of lymphocytes significantly compared bias (Fig. 5).
with the model controls (SMD:-4.37, 95% CI:
6.84, 1.90, P ¼ 0.0005, I2 ¼ 86%) (Fig. 4).
4 Discussion
Total Cells Of the 13 included studies,
5 measured the number of total cells in BALF
4.1 Overview
(Aimbire et al. 2008, da Cunha Moraes et al.
2018, Fazza et al. 2020, Miranda da Silva et al.
Our meta-analysis of present evidences may shed
2015, Oliveira Jr. et al. 2014), which were
light on the protective, anti-inflammatory, and
included in the meta-analysis. The analysis
anti-oxidative effects of PBM therapy in lung
revealed that PBM therapy reduced the number
injury-induced animal model with especial focus
of total cells significantly compared with the
on COVID-19. Human experience in experimen-
model controls (SMD:-3.54, 95% CI: 5.30,
tal and animal models of lung disease, ARDS,
1.79, P < 0.0001, I2 ¼ 84%) (Fig. 4).
38 H. Raji et al.
Fig. 4 Comparison of the effect on BALF cells between CIs for each study. SMD standardized mean difference, CI
PBM therapy and control in the animal lung injury models. confidence interval, PBM photobiomodulation, SD stan-
The figure represents the SMD result of the overall exper- dard deviation, IV independent variable
imental data, and the horizontal lines represent the 95%
and other infections has shown that PBMT has inflammatory cytokine. Studies have shown that
cellular and molecular effects at several levels the PBM can increase the IL-10 generation and
against cytokine storms (Jahani Sherafat et al. improve the balance of inflammatory processes.
2020). PBMT reduces pro-inflammatory Our pooled analysis also showed that PBMT
interleukins (IL-1β, IL-6 levels, MIP-2, mRNA re-regulates anti-inflammatory cytokines such as
expression TNF-α, etc.) (Cardoso et al. 2020). In IL-10, which lead in an increase in IL-10 values.
this context our meta-analysis also showed the IL-10 is an immune-activating cytokine that plays
significant beneficial effect of PBMT in reducing a fundamental role in restraining host immune
IL-1β, IL-6, and TNF-α. IL-10 is an anti- response against pathogen infections; thus,
inflammatory cytokine which can reduce the tis- dysregulation of IL-10 is accompanied with
sue injury with adjusting the production of other greater immunopathological response to infection
The Probable Protective Effect of Photobiomodulation on the Inflammation of. . . 39
Fig. 5 Funnel plot analysis of variables of interest. The standardized mean difference is plotted against the standard error
of the standardized mean difference. A, different cytokines in lung tissue; B, different BALF cells
along with more risk of developing many autoim- cytokine pathways in patients with COVID-19
mune diseases (Iyer and Cheng 2012) due to the (Dhar et al. 2021).
fact that IL-10 levels increase in inflammatory PBMT reduces pulmonary vascular leakage
diseases; therefore, there are many therapeutic and activates macrophages, T cells, and neutro-
opportunities for interventions through this phil infiltration. The P2X7 receptor (P2X7r) is
40 H. Raji et al.
recognized as a potential new therapeutic target in and treating such diseases (Danlos et al. 2021,
the pathogenesis of COVID-19 (Pacheco and Feldmann et al. 2020). IL-6 is a pleiotropic cyto-
Faria 2021). P2X7r is primarily expressed in kine and has a key role in ARDS pathophysiol-
many cells and a major factor in the activation ogy, of which higher levels of IL-6 in the lung and
of cytokine storms and lung pathology in plasma are related to the poor prognosis of the
response to viruses. PBMT regulates P2X7r disease (Goldman et al. 2014). PBMT can
expression and reduces collagen deposition. Due decrease the IL-6 levels during the acute lung
to the benefits of PBMT and the lack of inflammation or ARDS, especially in patients
established treatments for COVID-19 diseases, it with COVID-19 (Jahani Sherafat et al. 2020).
seems that PBMT can be useful in controlling Besides, IL-1β is the main inflammatory cytokine
COVID-19 as an alternative or preventative treat- to the initiation of inflammation and causes poor
ment, especially in severe cases. According to our prognosis in ARDS patients (Meduri et al. 1995).
pooling results, TNF-α was decreased by PBMT, Neutrophils are the main source of this cytokine
in which this reduction could be due to decrease (Tecchio et al. 2014). It has been found that the
in the mRNA expression of TNF-α and its pro- PBMT can reduce the production of this cytokine.
duction level (Saxena et al. 2020). This change in
the mRNA expression of TNF-α in viral
infections has been reported previously (Fang 4.2 Clinical Implications
et al. 2003). TNF-α activates neutrophil adhesion
and IL-6 generation and can increase the coagula- Using search terms through selected databases,
tion and edema in the acute lung injury (Kany there were only five studies on using PBM ther-
et al. 2019). Although normal levels of TNF-α are apy in patients with COVID-19 (Table 2). Of five
important for the regulation and persistence of studies, only one clinical trial included PBM ther-
immune responses to its overproduction which apy to treat COVID-19, and the rest four were
can lead to some inflammatory or autoimmune either case report or case series (Pelletier-
diseases, especially COVID-19, therefore, Aouizerate and Zivic 2021, Sigman et al.
neutralizing TNF-α or blocking its receptors can 2020a, b, Teixeira et al. 2021, Vetrici et al.
be an effective therapeutic strategy in controlling 2021) (Table 3).
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Adv Exp Med Biol - Cell Biology and Translational Medicine (2022) 15: 45–59
https://doi.org/10.1007/5584_2021_674
# Springer Nature Switzerland AG 2021
Published online: 5 November 2021
B. Arjmand (*)
Cell Therapy and Regenerative Medicine Research Center, M. Rezaei-Tavirani
Endocrinology and Metabolism Molecular-Cellular Proteomics Research Center, Shahid Beheshti University
Sciences Institute, Tehran University of Medical Sciences, of Medical Sciences, Tehran, Iran
Tehran, Iran e-mail: Tavirany@yahoo.com
Metabolomics and Genomics Research Center, P. Goodarzi and F. Mohamadi-Jahani
Endocrinology and Metabolism Molecular-Cellular Brain and Spinal Cord Injury Research Center,
Sciences Institute, Tehran University of Medical Sciences, Neuroscience Institute, Tehran University of Medical
Tehran, Iran Sciences, Tehran, Iran
e-mail: barjmand@sina.tums.ac.ir e-mail: pr_goodarzi@yahoo.com;
f.mohamadijahani@gmail.com
S. Alavi-Moghadam and A. Tayanloo-Beik
Cell Therapy and Regenerative Medicine Research Center, N. Mehrdad
Endocrinology and Metabolism Molecular-Cellular Elderly Health Research Center, Endocrinology and
Sciences Institute, Tehran University of Medical Sciences, Metabolism Population Sciences Institute, Tehran
Tehran, Iran University of Medical Sciences, Tehran, Iran
e-mail: sepidalavi@gmail.com; a.tayanloo@gmail.com e-mail: emri-research@tums.ac.ir
P. Parhizkar-Roudsari B. Larijani (*)
Metabolomics and Genomics Research Center, Endocrinology and Metabolism Research Center,
Endocrinology and Metabolism Molecular-Cellular Endocrinology and Metabolism Clinical Sciences
Sciences Institute, Tehran University of Medical Sciences, Institute, Tehran University of Medical sciences, Tehran,
Tehran, Iran Iran
e-mail: Peyvand.parhizkar@yahoo.com e-mail: emrc@tums.ac.ir
45
46 B. Arjmand et al.
lineage. In this context, they have developed to membrane glycoprotein (M) (Schoeman and
modify many pathways in host cells (Gelderblom Fielding 2019; Li et al. 2020a, b, c). The first
1996; Chiu et al. 1997; Lodish et al. 2000). Herein, step in the pathogenicity of SARS-CoV-2 is the
in recent years, omics investigations have shown binding of coronavirus proteins to the host-cell
that viruses can lead to large-scale modifications in special surface receptor named angiotensin-
the host cell’s metabolic pathways in accordance converting enzyme 2 (ACE2), which leads to
with their own particular needs. Moreover, studies the membrane fusion and paves the way for the
have shown that different species of viruses may virus to enter the host cell (Ni et al. 2020a, b;
alter specific metabolic pathways in the host cell Shang et al. 2020; Yesudhas et al. 2020; Zhang
(Sanchez and Lagunoff 2015a, b; Thaker et al. et al. 2020a, b). In this respect, the expression and
2019; Martín-Vicente et al. 2020). Hereupon, distribution of entrance receptors subsequently
understanding the metabolic pathways involved impact viral tropism and pathogenicity
in the viral infection process can lead to the emer- (Mansfield 2007; Maginnis 2018; Harrison et al.
gence of innovative therapeutic strategies through 2020). In general, the life cycle of the virus after
targeted repression of special metabolic pathways entering the host cell includes escaping the
(Mayer et al. 2019; Purdy 2019; Keshavarz et al. immune response of the host cell and replicating
2020). Currently, the most recent type of viral the virus by regulating the machinery of the host
infection that has begun in Wuhan, China, in late cell for genome replication and protein synthesis
December of 2019 and involved innumerable peo- (Lucas et al. 2001; Rampersad and Tennant 2018;
ple around the world is coronavirus disease Zhang et al. 2020a, b). Accordingly, viral and
(COVID-19 disease), which was caused by a host metabolic processes are closely related, and
new type of coronavirus called the novel 2019 changes in host metabolism occur during infec-
coronavirus (2019-nCoV) or severe acute respira- tion at all levels – cellular, tissue, organ, and
tory syndrome (SARS) coronavirus 2 (SARS- physiological (Arnold et al. 2013; Ayres
CoV-2) (Alavi-Moghaddam 2020; Arjmand et al. 2020a, b; Troha and Ayres 2020). SARS-CoV-
2020; Azodi et al. 2020; Roudsari et al. 2020). 2 infection as a multisystem condition can lead to
Accordingly, the authors of the present review mild to severe illness with different symptoms.
have attempted to discuss the metabolomics per- The majority with the infection undergoes a mild
spective in COVID-19. form of the disease, while a subset of individuals
reveals a severe or critical form (Funk and
Ardakani 2020; Organization, W. H 2020;
2 Novel Corona Viral Infection Roudsari et al. 2020). Herein, the host’s reaction
to the infection determines the type of involve-
SARS-CoV-2 (a member of the Coronaviridae ment, which in severe and critical cases, espe-
family within the Nidovirales order) as a highly cially in subjects with comorbidities such as
infectious and pathogenic virus has triggered a severe obesity, diabetes, chronic obstructive pul-
pandemic acute respiratory disease, and monary disease, hypertension, cardiovascular dis-
according to World Health Organization (WHO) ease, etc., may lead to dysfunction and damaging
declaration, it threatens public health and safety of several systems (Basu et al. 2020; Sanyaolu
(Sharma et al. 2020; Weill et al. 2020). The struc- et al. 2020). Furthermore, many of the
ture of the SARS-CoV-2 with crown-like appear- pathogeneses found in COVID-19 individuals
ance contains a single-stranded, positive-sense can be outcomes of various infectious condition
ribonucleic acid (RNA) genome which is collections that developed to critical grades, and
surrounded by a lipid bilayer membrane. Each they are not necessarily among novel infectious
particle of the virus is approximately 50–200 nm conditions (Cevik et al. 2020; of the International,
in diameter. In addition, it has four structural C. S. G 2020; Shahriarirad et al. 2020).
proteins called spike glycoprotein (S), nucleocap- Immune System Activation in Response to
sid protein (N), envelope glycoprotein (E), and Novel Coronavirus 2019 The immune system of
48 B. Arjmand et al.
the hosts normally can support the body’s natural factors and immune activation due to direct viral
ability to protect against viruses and diseases effects, downregulation of ACE2 after COVID-
along with the maintaining tissue homeostasis. 19 infection can cause that imbalance between
Moreover, it can deliver antibodies to eliminate renin-angiotensin system (RAS) and ACE2/
pathogens (Rouse and Sehrawat 2010; Parham angiotensin-(1–7)/MAS axis leading to lung
2014; Thakur et al. 2019). In this context, injuries and other organ’s damages (Ni et al.
investigations have reported that the metabolic 2020a, b; Pinto et al. 2020; Rivellese and
pathways can amplify the effector capacities of Prediletto 2020), because inhibited ACE2 and
immune cells by providing sufficient energy activated ACE1 make angiotensin II act through
(adenosine triphosphate (ATP)) and metabolic angiotensin-1 receptor (AT1R) or AT2R and pro-
intermediates. Further, metabolic reprogramming vide pro-inflammatory responses in addition to
can be effective to determine immune replies stimulated aldosterone secretion. These processes
(Pearce and Pearce 2013; Ganeshan and Chawla can lead to hypokalemia and higher blood pres-
2014; Domblides et al. 2018). In the case of sure. The risk of breathing disorder also increases
SARS-CoV-2 infection, it can be mediated by T due to the higher vascular permeability locally
lymphocyte activation and production of inflam- (Fig. 1) (Bornstein et al. 2020). Herein, it has
matory mediators (e.g., tumor necrosis factor-beta been also confirmed that angiotensin 1–7 acting
(TNF-β), interleukin (IL)-6, IL-1, type I inter- through Mas receptor has anti-inflammatory
feron (IFN-I), and CCL2) as well as generation effects and anti-fibrotic responses along with its
of perforin and granzyme B (Guidotti and Chisari inhibitory influences on vascular and cell growth
2000; Chen et al. 2010; Chowdhury et al. 2020; mechanisms (Simões e Silva et al. 2013). Here-
Oliveira et al. 2020; Lewis-Wade 2020). upon, the imbalance between these pathways
leading to increased activation of AT1R and
also AT2R can be the case in hypertension.
3 Metabolic Pathways in Immune Besides the existing coordination between
System Activation SARS-CoV-2 and hypertension, there is a proba-
ble link to type 2 diabetes mellitus (T2DM) and
There are several metabolic pathways that have metabolic syndrome, because of the existence of
roles in peripheral blood and bronchioalveolar metabolic inflammation which predisposes
lavage fluid of COVID-19 patients (Ayres coronavirus-infected patients to a higher release
2020a, b; Gardinassi et al. 2020). Indeed, as men- of cytokines. Thus, the coordination of T2DM
tioned, SARS-CoV-2 enters the host cell using and coronavirus can lead to more serious and
ACE2 as the receptor in different organs and long-lasting lung pathology because of
tissues. Viral spike glycoprotein activation and dysregulated immune responses. Also, the coro-
cleaving the C-terminal part of ACE2 (via trans- navirus can bind to its receptor, ACE2, in the
membrane protease serine 2 (TMPRSS2) and pancreas and results in β-cell dysfunction, dam-
FURIN proteases) make this entry more facile aged islets, and reduced insulin release. This pro-
(Fig. 1) (Ni et al. 2020a, b). Herein, it can be cess can lead to hyperglycemia and transient
stated that SARS-CoV-2 infection leads to the T2DM (Bornstein et al. 2020). On the other
higher secretion of cytokines including IL-6 and hand, it has been explained that increased expres-
tumor necrosis factor-α (TNF-α) through the sion of genes that have roles in metabolic
effects on ACE2 receptor. It can subsequently pathways such as heme biosynthesis, oxidative
cause the higher levels of acute-phase proteins phosphorylation, and tryptophan metabolism
such as serum amyloid P-component (SAP), may be linked to COVID-19 infection
C-reactive protein (CRP), serum amyloid (Gardinassi et al. 2020). Hereupon, higher expres-
(SA)A1, SAA2, and C6 which seem to be con- sion of genes linked to heme-hemoglobin metab-
siderably elevated in the severe group of patients olism pathway has been found that might affect
(Shen et al. 2020). Besides the inflammatory sepsis secondary to pneumonia. It has been found
Metabolomics Signatures of SARS-CoV-2 Infection 49
Fig. 1 A view of ACE2 receptor regulation and effects MAS axis (Ni et al. 2020a, b; Pinto et al. 2020; Rivellese
in COVID-19 infection. Spike glycoprotein activation of and Prediletto 2020); hypokalemia; and higher blood pres-
the virus along with the effects of TMPRSS2 and FURIN sure are some of the probable effects of this viral entry
proteases on ACE2 makes viral entry more facile (Ni et al. (Bornstein et al. 2020). ACE2, angiotensin-converting
2020a, b). The higher secretion of cytokines; increased enzyme 2; TMPRSS2, transmembrane protease serine 2;
levels of acute-phase proteins such as SAP, CRP, SAA1, SAP, serum amyloid P-component; CRP, C-reactive pro-
SAA2, and C6 (Shen et al. 2020); imbalanced renin- tein; SA, serum amyloid; RAS, renin-angiotensin system
angiotensin system (RAS) and ACE2/angiotensin-(1–7)/
unable to fulfill the hypermetabolic demands of 2020). In the next parts, more detailed effects of
COVID-19 sepsis; thereafter mitochondrial the virus on host metabolism will be stated.
proteins can be presented as damage-associated
molecular pattern (DAMP) that activates innate
immunity. It can reveal the negative effect of 4.1 Carbon Metabolism
aging on sepsis, too (Shenoy 2020). The modula-
tion of tricarboxylic acid (TCA) cycle, mannose, It has been clearly known that in order to access
fructose, carbon, lipid, nucleotide, protein, and substantial levels of ATP for biological pathways,
galactose metabolism in addition to glycolysis glucose utilizes aerobic metabolism. In aerobic
and gluconeogenesis has been also reported in conditions, it can also go through pentose phos-
mass spectrometry measurements and can under- phate pathway and generates nicotinamide ade-
lie the metabolic pathways of the disease nine dinucleotide phosphate (NADPH). As the
(Gardinassi et al. 2020; Shen et al. 2020). The result, the appropriate ratio of oxidized glutathi-
effects of novel coronavirus 2019 on switching one (GSH) to glutathione is provided that take
host metabolism of carbon, lipids, nucleic acid, parts in antioxidant defense system and helps in
and proteins are going to be explained in more destroying pathogenic microorganisms beside
details in the next parts. immune system (Nelson and Cox 2017). Inflam-
matory conditions make immune cells switch
their mitochondrial oxidative phosphorylation to
4 Switching Host Metabolism by cytosolic aerobic glycolysis according to the
Novel Coronavirus 2019 Warburg effect. Some factors and proteins such
as HIF-1α, mammalian target of rapamycin
Some molecular changes have been found in the (mTOR), and serine/threonine kinase have roles
sera of patients with COVID-19 such as in this alteration that make immune cells more
dysregulation of macrophage, degranulation of phagocytic. This switch also accelerates ATP
platelets, complement system involvement, and production and provides a massive amount of
massive suppression of metabolic condition that metabolic precursors for higher proliferation and
prove the considerable alteration of protein and more cytokine release (Reiter et al. 2020a, b).
metabolite status in patients (Sanchez and Therefore, aerobic glycolysis may lead to cyto-
Lagunoff 2015a, b; Shen et al. 2020). COVID- kine storm that in association with increased oxi-
19 patient subjects have also shown an unusual dative stress can result in pneumonia in COVID-
rise in some biochemical parameters such as 19 subjects (Reiter et al. 2020a, b). It was also
serum ferritin, erythrocyte sedimentation rate, found that pyruvate cannot convert into acetyl-
CRP, albumin, and also lactate dehydrogenase coenzyme A, when mitochondria are adopted to
that may underlie the ability of SARS-CoV-2 to aerobic glycolysis. This results in a lack of avail-
change the host’s metabolism (Singh et al. 2020). able mitochondrial melatonin which has a signifi-
Viral genome replication through altering host cant antioxidant and a potent anti-inflammatory
nucleotide metabolism and virion assembly by activity (Reiter et al. 2020a, b). On the other hand,
assessing host amino acids are some of the under anaerobic conditions (in COVID-19
benefits of their ability for changing host metabo- infected subjects), glycolysis pathway makes the
lism. They also assess carbohydrates to provide pyruvate be generated from glucose. Then pyru-
their energy source and fatty acids to form viral vate is fermented to lactate that provides an insuf-
envelope in addition to glycoprotein involvement. ficient amount of ATP (Nelson and Cox 2017; Li
In this regard, it can be understood that the et al. 2020a, b, c). SARS-CoV-2 utilizes massive
host’s metabolic status, medical/environmental ATP levels and induces the anaerobic glycolysis
conditions, age, and sex have important process which forms increased levels of lactate.
influences on the prognosis of the SARS-CoV- Continuously, hypoxia and insufficient ATP pro-
2 infection (Gasmi et al. 2020; Soliman et al. duction can lead to higher amounts of blood
Metabolomics Signatures of SARS-CoV-2 Infection 51
lactate and lactate dehydrogenase (LDH) because host lipid profile. Additionally, they can specifi-
of unsuccessful lactate metabolism for gluconeo- cally accumulate different types of lipids such as
genesis or oxidization via citric acid cycle. ATP glycerophospholipids, sphingolipids, and sterols
depletion condition can affect all of the pathways in VRCs (Strating and van Kuppeveld 2017). In
that utilize ATP, for instance, glucose uptake and several studies, the association of COVID-19
pentose phosphate pathway in addition to oxida- infection with dyslipidemia has been reported;
tive decarboxylation of pyruvate and citric acid for instance, reduced serum high-density lipopro-
cycle. Insufficient ATP production also induces tein (HDL) cholesterol level in early stages of the
the production of glucose through the decompo- disease can be seen especially in severely infected
sition of hepatic glycogen. Persistent hypoxia can patients. Thus, declined HDL cholesterol concen-
result in hyperglycemia condition, because this tration might have an association with the severity
glucose has not the ability to convert into fatty of COVID-19 (Hu et al. 2020; Li et al.
acids or amino acids. On the other hand, blocked 2020a, b, c). Metabolomics profiling of COVID-
pentose phosphate pathway results in lower glu- 19 sera of patients has shown totally
tathione levels causing impaired balance between downregulation of over 100 lipids (sphingolipids,
the oxidative and antioxidative status of the body. glycerophospholipid, and fatty acids). Herein,
Thus, it leads to the lower ability for attenuating dysregulation of several apolipoproteins (APO)
oxidative impairments that are accelerated in such as APOA1, APOA2, APOH, APOL1,
COVID-19 subjects (Li et al. 2020a, b, c). More- APOD, and APOM has been also found that
over, in the metabolomics researches, higher mostly was in association with macrophage
levels of glucose and glucuronate have been also activities and were downregulated. In addition,
found that along with elevated bilirubin degrada- metabolites which have roles in lipid metabolism
tion product, four bile acid derivatives can show were dysregulated, too. Moreover, steroid
the declined detoxification activity of the liver too hormones have been accumulated in COVID-19
(Shen et al. 2020). infected subjects that may be linked to increased
macrophage function. Decreased sphingolipid
levels have been also seen in non-severe and
4.2 Lipid Metabolism severe patients, and it has been known that
biomembranes consist of sphingolipids and
Viruses can attack synthesis and signaling of the glycerophospholipids that are important for the
lipids and alter the host cells in order to provide activation of immune processes. Sphingolipids
lipids for their envelopes, because lipids have a also have roles in different cellular and inflamma-
great involvement in envelopment in addition to tory processes. It has been found that sphingo-
membrane fusion and transformation that are sine-1-phosphate can promote macrophage
important for viral replication (Abu-Farha et al. activation and induce their migration to inflam-
2020). Thus, lipids have crucial roles in diverse matory sites (Shen et al. 2020). On the other
stages of the virus life cycle and lipid metabolism hand, it should be mentioned that switching host
pathway and can affect viral propagation. Herein, metabolism to fasting state can result in higher
lipids play their functions as direct receptors for levels of ketones, higher mitochondrial stress
viruses in addition to their effects on forming the resistance, increased antioxidant defenses, and
viral replication complex. Lipids can also provide increased autophagy. It can also lead to more
the energy needed for viral replication (Yan et al. DNA repair and decreased protein synthesis.
2019). Positive-strand RNA viruses have the abil- Thus, changes in the lipid metabolism of host
ity for remodeling cellular membrane in order to are important to note and should be considered
generate viral replication compartments (VRCs) as therapeutic targets (Soliman et al. 2020).
that are the sites for the replication of viral RNA. About the effects of COVID-19 infection on the
It reveals that viruses have great effects on lipid host’s lipid metabolism, it could be also stated
metabolism along with diverse interactions with that recovered patients may experience higher fat
52 B. Arjmand et al.
(Gordon et al. 2020). It has been found that amino 6 Therapeutic Suggestions
acid residues 111–158 (related to S protein beta Targeting Metabolic Pathways
coronavirus) have interactions with sialic acids
(on the gangliosides of host cells) (Thomas et al. Metabolic processes of COVID-19 infection are
2020). In addition to the massive interaction tightly linked to the disease pathogenesis; thus,
found between coronavirus and human proteins, targeting metabolic pathways at different levels
a couple of small protein complexes have been can result in improved defense ability against
also mentioned that were specifically relevant to COVID-19 at its different stages (Ayres
coronavirus infection; EIF4E2-GIGYF2 dimer, 2020a, b) (Fig. 2). Therefore, although COVID-
MAT2A-MAT2B complex, DNA-PK kinase, 19 is not a metabolic disease in the first place, the
and the mitochondrial proteins (including PHB, metabolic control of the disease is an essential
PHB2, and STOML2) are the noted complexes therapeutic approach for it. Herein, metabolic
which act on the repression of protein translation, control of T2DM and coordinated parameters
controlling S-adenosyl methionine (SAM) syn- such as glucose, blood pressure, and lipid levels
thesis, contributing to interferon induction, and is important in order to reduce severe
regulating mitophagy in order (Perrin-Cocon complications and also to help for treating
et al. 2020). patients (Bornstein et al. 2020). Glucagon-like
peptide-1 (GLP-1) agonists as a class of
antidiabetic medications have positive effects on
5 The Importance glucose metabolism and also blood pressure.
of Metabolomics They also prevent coronavirus from entering the
in Investigating Viral-Host host cell. Their influences on better metabolic
Interactions pathways and also induction on the activity of
protective ACE2/Mas receptor activity may lead
Metabolomics (as an analytical profiling tech- to improved pulmonary function (Simões e Silva
nique and “omics” technologies) can quantify et al. 2013). Moreover, in order to induce and
and compare large numbers of metabolites pres- reactivate the ACE2 system in COVID-19
ent in biological samples and provides a window infected patients, angiotensin-converting enzyme
on metabolic pathways using high-throughput inhibitors (ACEIs), angiotensin II receptor
analytical chemistry and analysis of multivariate blockers (ARBs), and recombinant ACE2 may
results (Agharezaee et al. 2018; Rahim et al. have prophylactics and therapeutic benefits
2018; Arjmand 2019a, b; Gilany et al. 2019; (Chatterjee and Thakur 2020; Lei et al. 2020).
Goodarzi et al. 2019; Larijani et al. 2019; Also, polymorphisms in ACE2 or TMPRSS2
Mehrparavar et al. 2019; Tayanloo-Beik et al. DNA have shown a considerable association of
2020). Moreover, an idea of the disease severity genetic susceptibility of the disease that guides
along with the predictive outcome of the viral effective and personalized treatments including
infection can be given by metabolite profiling hydroxychloroquine and camostat. This light up
or metabolomics assays. In other words, the benefit of host genetic initiative for COVID-
metabolomics is applied to review the impress 19 (Hou et al. 2020). Moreover, because of the
of host-virus interaction at the metabolic level or important effects of host cell proteases to activate
the impacts of viruses on host cell metabolism envelope glycoproteins and thus, viral entry, they
(Voge et al. 2016; Blasco et al. 2020; Roberts can be also beneficial targets for antiviral inter-
et al. 2020). Indeed, metabolomics can help to vention (Arakelyan 2020). Homocysteine also is
provide appropriate treatment approaches via able to activate angiotensin II (type I) receptor;
identifying unique metabolic perturbations cre- thus, homocysteine-mediated transsulfuration
ated by viruses (Zhang et al. 2015; Byers et al. system, vitamin B6, vitamin B12, and folic acid
2019). should be considered in treatment approaches
54 B. Arjmand et al.
Antidiabetic
medications ACEIs,
Modulating
sirtuin ARBs,
activity recombinant
ACE2
Arbidol Homocysteine
LJ-001 mediated
hydroxychloro
-quine
trans
methyl-β- -sulfuration
Some of The system
cyclodextrin Therapeutic
Suggestions
Targeting
Metabolic Pathways Hb−HO-1
Statins
system-
fibrate
targeted
AM580
drugs
Melatonin Oxygen
therapy therapy
Regular
blood glucose
controlling
Fig. 2 Therapeutic suggestions targeting metabolic drugs (Maiti 2020), oxygen therapy, regular blood glucose
pathways. Some of the promising therapeutic approaches controlling (Li et al. 2020a, b, c), melatonin therapy
for COVID-19 targeting metabolic pathways are (Reiter et al. 2020a, b), statins, fibrate, AM580, arbidol,
concluded in this figure including antidiabetic medications LJ-001 hydroxychloroquine, methyl-β-cyclodextrin
(Simões e Silva et al. 2013), ACEIs, ARBs, recombinant (Abu-Farha et al. 2020), and modulating sirtuin activity
ACE2 (Chatterjee and Thakur 2020, Lei et al. 2020), (Shenoy 2020). ACEIs, Angiotensin-converting enzyme
homocysteine-mediated transsulfuration system-targeted inhibitors; ARBs, angiotensin II receptor blockers
drugs (Singh et al. 2020), Hb-HO-1 system-targeted
(Singh et al. 2020). On the other hand, as hemo- apoptotic effects that in addition to iron chelation
globin dysfunction may lead to decreased could be another promising target for the disease
oxygenation, higher free iron level, and (Maiti 2020). According to negative effects of
downregulated heme oxygenase-1 (HO-1), the hypoxia, benefits of oxygen intake (Handy et al.
Hb-HO-1 system is mentioned as a suggested 2005), and what was mentioned about unbalanced
therapeutic target for COVID-19 (Maiti 2020). It glucose aerobic metabolism during COVID-19, it
is stated that CO (a metabolite of HO-1) levels is also important to correct this condition using
besides endogenous HO-1 are in correlation with oxygen therapy in addition to regular blood glu-
the severity of COVID-19 (Donnay 2020). More- cose controlling (with test strips) (Li et al.
over, CO has anti-inflammatory and anti- 2020a, b, c). The use of melatonin with its anti-
Metabolomics Signatures of SARS-CoV-2 Infection 55
Declaration The authors declare that they have no con- Loney C, Thai J, Miller ZD, Lin AE, Schmidt MM,
flict of interest. Stewart DG, Goldfarb D, De Lorenzo G, Rihn SJ,
Voorhees RM, Botten JW, Majumdar D, Guttman M
(2020) SARS-CoV-2 disrupts splicing, translation, and
Ethical Approval This article does not contain any stud-
protein trafficking to suppress host defenses. Cell
ies with human participants or animals performed by any
Basu M, Saha C, Choudhury KR, Dutta S, Ghosh S,
of the authors.
Chowdhury S, Mukhopadhyay S, Bhattacharyya NP
(2020) COVID-19 comorbidity and metabolic syn-
Authors’ Contributions All authors contributed to the drome: is there a Molecular basis?
study conception and design. Sepideh Alavi-Moghadam and Belladonna ML, Orabona C (2020) Potential benefits of
Peyvand Parhizkar Roudsari wrote the first draft. Akram tryptophan metabolism to the efficacy of tocilizumab in
Tayanloo-Beik, Mostafa Rezaei-Tavirani, and Fereshteh COVID-19. Front Pharmacol 11(959)
Mohamadi-jahani helped to study and gather information. Biancatelli RMLC, Berrill M, Mohammed YH, Marik PE
Parisa Goodarzi and Neda Mehrdad extensively edited the (2020) Melatonin for the treatment of sepsis: the scien-
manuscript. Bagher Larijani participated in a critical review. tific rationale. J Thorac Dis 12(Suppl 1):S54
Babak Arjmand helped supervise the project and gave final Blasco H, Bessy C, Plantier L, Lefevre A, Piver E,
approval of the version to be published. Bernard L, Marlet J, Stefic K, de Bretagne IB, Cannet
P (2020) The specific metabolome profiling of patients
infected by SARS-COV-2 supports the key role of
tryptophan-nicotinamide pathway and cytosine
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https://doi.org/10.1007/5584_2021_666
# Springer Nature Switzerland AG 2021
Published online: 5 September 2021
61
62 D. Agas and M. G. Sabbieti
Fig. 1 Representative images of sub-metaphyseal and and mature cells (perivascular niche). Magnification 20x.
epiphyseal bone marrow areas from mice femur. (a, b) (c, d) Bone marrow endosteal (c) and perivascular (d)
Histological section of the bone marrow stained with tolu- region stained with hematoxylin/eosin stain. Magnifica-
idine blue. (a) Note the close proximity of bone marrow tion 40x. Slides were imaged using a Zeiss Axioplan
elements near to endosteum (endosteal niche). (b) Central fluorescence microscope. CB cortical bone, S sinusoids,
diaphyseal bone marrow region occupied by arterioles and CV central vein
sinusoids, which remain surrounded by stem, progenitor,
In sum, current research is achieving a better next to the perivascular area (Ellis et al. 2011).
understanding of the bone marrow by identifying The vascular niche encompasses the sinusoidal
and characterizing spatiotemporal stem and and arteriolar vessels, bordered by MSCs, HSCs,
mature cell affinities and further defining niche lineage-committed progenitors, and mature cells.
topographical features. The sinusoids are lined with endothelial cells in
the inner part and by adventitial reticular cells in
the outer part, providing a proper perivascular
2 The Bone Marrow Niche space for HSC adhesion and retention. Indeed,
Topography sinusoidal endothelial cells facilitate HSC hom-
ing through the release of type IV collagen,
Although the endosteum can be as far as five cells integrins, selectins, and cytokines, while adventi-
away from the bone marrow vasculature (Kiel tial reticular cells further contribute to HSC reten-
et al. 2007), about 15% of the CD150+CD48 tion within the bone marrow via the release of
HSCs fraction resides within the endosteal com- CXCL12 and c-kit ligand (stem cell factor) (Lai
partment, while about 60% of this subset is found et al. 2014; Czechowicz et al. 2007). In
64 D. Agas and M. G. Sabbieti
counterpart, higher levels of reactive oxygen spe- GFP+c-kit+ non-dividing HSCs were mainly
cies (ROS) near sinusoidal endothelial cells can localized near sinusoids, while Ki67+α-catulin-
enhance stem cell differentiation and mobiliza- GFP+c-kit+ dividing HSCs were located near the
tion, while at the level of arterial endothelial bone surface (Acar et al. 2015). Another report
cells, the formation of a low-ROS (ROSlow) indicated that arterioles covered exclusively by
microenvironment improves HSC quiescence rare NG2+ pericytes create an optimal microenvi-
and self-renewal (Ito et al. 2006). Blood vessel ronment for HSC quiescence; the dormant HSCs
permeability is directly related to increased blood were detected near the Sca-1+ arterioles and arte-
plasma ROS levels, which facilitate HSC migra- riolar NG2+ pericytes, in contrast to the cycling
tion capacity while reducing their long-term Ki67+ HSC subset, which was located next to
repopulation and quiescent potential (Itkin et al. sinusoid-associated leptin receptor (LepR+) cells
2016). HSCs surrounding sinusoidal endothelial (Kunisaki et al. 2013). Furthermore, NG2+ deple-
cells (marked as Sca-1 ) and located more than tion destabilizes arteriolar HSC niches and drives
20 μm from bone marrow arterial endothelial cells HSCs to a non-quiescent status (Kunisaki et al.
(marked as Sca-1+) can deal with either low or 2013).
high ROS levels, in contrast to arterial HSCs, The widespread presence of the adherence and
which experience a constant ROSlow background. tight junction mediators VE-cadherin and ZO-1
Specifically, HSC, located within defined ROSlow mainly on Sca-1+ arterial bone marrow endothe-
areas distant from arteries, cohabitates next to lial cells (compared to sinusoids) further supports
megakaryocytes, establishing a peculiar quiescent the effectiveness of this ROSlow compartment for
megakaryocytic stem cell niche (Itkin et al. 2016). HSC maintenance (Itkin et al. 2016). At inter- and
Indeed, 20.8% of (Lin) CD41 CD48 CD150+ outer-sinusoidal level, the extracellular matrix
cells have been found in direct contact with formed by a specialized laminin reticular fiber
bone marrow megakaryocytes and 48.6% of the network (with laminin 421 as the main compo-
same HSC lineage at a distance of two cells from nent of murine marrow) is thought to enhance
them (Zhao et al. 2014). The spatial vicinity HSC and progenitor cell cycling. Laminin α4
suggests an operational relationship between ablation blocks Lin c-kit+Sca+CD48 LT- and
megakaryocytes and HSCs and thus is thought ST-HSC cycling at the G0 phase and puts
to indicate megakaryocyte-mediated maintenance HSPCs in a quiescent cell cycle. Moreover, in
of HSC quiescence. Megakaryocyte ablation Lama4 / bone marrow, researchers observed
triggers quiescent HSC activation with concurrent reduced HSC homing and impaired HSC recircu-
HSC expansion (Zhao et al. 2014). lation between the blood and bone marrow (Susek
Niche topographical features have always et al. 2018).
presented a difficult knot to untangle, and In line with these observations, a study on
researchers have sometimes obtained differing Hoxb5-mCherry mice detected 94% of Hoxb5+
results depending on the explicit stem cell HSC homing next to endothelial cells, where they
sub-population and its specific spatiotemporal establish a peculiar niche for LT-HSC lodging
commitment and needs. A deep-imaging study (Chen et al. 2016). In addition, the H-type endo-
of the distribution of α-catulin-GFP+c-kit+ cells thelium, a specific section of arteries and capillary
indicated that HSCs populate predominantly the endothelial cells, is able to release critical HSC
diaphyseal central bone marrow area, next to maintenance factors within the bone marrow res-
leptin receptor+ and Cxcl12high niche cells, rather ervoir (Kusumbe et al. 2016). Current findings
than the bone endosteal surface. Interestingly, in based on Mds1GFP/+Flt3Cre mice unearthed a
catulinGFP/+ mice, 85% of α-catulin-GFP+c-kit+ novel LT-HSC population (termed MFG cells)
HSCs reside in a radius of 10 μm around the that accounts for 12% of the total LT-HSCs and
perisinusoidal area but remain apart from represents a quiescent bone marrow HSC fraction
arterioles and transition zone capillaries (Acar with potent repopulation potential. Both MDS1-
et al. 2015). Fully 81% of Ki67 α-catulin- GFP HSPC and MFG-HSC subsets were found
Autophagic Mediators in Bone Marrow Niche Homeostasis 65
located within a range of 10 μm from the closest not exclusively via the release of cytokines/
vessel (Christodoulou et al. 2020). The most chemokines and physical cell-cell interactions
intriguing aspect of these findings is that while (Agas et al. 2015; Pinho and Frenette 2019;
MFG-HSCs are found exclusively perisinusoidal Crippa et al. 2019). Studies have reported dissim-
rather than peri-arteriolar, native MFG-cells are ilar MSC sub-populations with distinct opera-
also found close to the endosteum, highlighting tional commitments and distribution within the
the spatial/operational idiosyncrasy of these cells bone marrow. It is worth mentioning that the
within a cohesive endosteal-vascular niche sce- Nestin+ MSC subset provides angiopoietin-1
nario (Christodoulou et al. 2020). A further point (Ang-1), interleukin-7 (IL-7), vascular cell adhe-
to be noted is that hypoxia has been long consid- sion molecule 1 (VCAM1), stem cell factor
ered the main niche prerequisite for stem cell (SCF), and C–X–C motif chemokine ligand
quiescence (Takubo and Suda 2012). Although 12 (Cxcl12) for HSC maintenance (Mendez-
HSPCs and MFG-HSCs may be located far from Ferrer et al. 2010). Of note, Cxcl12 depletion
the highly hypoxic bone marrow micro-areas, from early Nestin LepR mesenchymal
they manage to maintain their dormant capacity progenitors significantly reduced HSC pool,
even in higher pO2 settings (pO2 > 10 mmHg) LT-HSC repopulating activity, and HSC quies-
(Christodoulou et al. 2020). cence (Greenbaum et al. 2013). Additional
Bone marrow niche homeostasis is strictly findings revealed that perisinusoidal Nestin-
related to the behavior of MSCs, since they GFPlow LepR+ cells can supply sinusoidal milieus
carry out critical functional tasks for HSC regula- with elevated levels of Cxcl12 and SCF, whereas
tion and bone dynamics. Actually, given the periarteriolar Nestin-GFPhigh NG2+ cells
capacity of MSCs for multilineage differentiation expressed predominantly high Cxcl12 levels
and self-renewal, it is thought that these (Kunisaki et al. 2013; Asada et al. 2017). In this
fibroblast-like bone marrow inhabitants have context, Cxcl12 deletion in LepR+ cells had no
stem cell properties, though there has long been significant effect on HSC dynamics; in contrast,
debate on their “stemness” potency. Therefore, deletion of Cxcl12 in NG2+ cells led to HSC
the scientific community has adopted a precise quiescence distress and HSC mobilization out of
line to distinguish the mesenchymal stem cells the arteriolar niche confines (Asada et al. 2017).
from the mesenchymal stromal cells within the In addition, these findings indicate the essential
bone marrow: “mesenchymal stem cell” indicates role played by arteriole-associated MSCs in
a stem cell population with multilineage differen- Cxcl12 synthesis for HSC maintenance and, as a
tiation and self-renewal ability, whereas “mesen- counterpart, that of LepR+ perisinusoidal cells as
chymal stromal cell” comprises the the foremost provider of SCF, indispensable for
heterogeneous plastic-adherent population HSC upkeep in-house (Asada et al. 2017).
applied mainly in tissue regenerating and immu- Perivascular LepR+ mesenchymal and endothelial
nomodulatory studies and in clinical trials cells have also been the subject of scrutiny, and
(Viswanathan et al. 2019; Nolta et al. 2020). one study suggested that perivascular endothe-
Thus, the MSC characterization used in this lium and Lepr-expressing cells constitute the
review corresponds to the mesenchymal/stromal major SCF source for HSC maintenance within
“trilineage” (osteoblasts, chondrocytes, and the vascular niche (Ding et al. 2012).
adipocytes) of pluripotent bone marrow Research on MSC subsets led to the identifica-
components. tion of CXC chemokine ligand (Cxcl)12-
MSCs have been depicted as abundant reticular (CAR) cells. CAR cells are
nonhematopoietic (CD45 ) and, based on their considered adipo-osteogenic progenitors and can
spatial distribution, nonvascular CD146+ or non- release high amounts of Cxcl12 and SCF for the
vascular Tie2 (Abarrategi et al. 2017). MSCs sustenance of HSCs and blood lineage
establish a fine-tuned regulatory network for progenitors. Specifically, CAR cells are essential
HSC maintenance within the niche, mainly but for blood cell development, and they provide
66 D. Agas and M. G. Sabbieti
support for HSC maintenance and for B cell and 2015). Intramural and paracrine signaling
erythroid progenitor expansion (Omatsu et al. molecules regulate expansion of stem cells and
2010). Perivascular CAR cells overlap with their differentiation fate. Notably, a vast number of
perisinusoidal LepR+ cells for Cxcl12 and SCF osteogenic factors, including BMP2, BMP7, and
assembly (Zhou et al. 2014), and thus both are Wnt3a, are also derived from the niche HSCs and
considered to be essential sources of HSC niche progenitor inhabitants (Chan et al. 2015).
factors. Recent work identified CAR/LepR+ cells, In addition, most colony-forming unit fibro-
a distinct MSC subset lodging near sinusoids and blast (CFU-F) activity has been attributed to a
endosteum that, through ablation of their adipo / perivascular PDGFRα+Sca-1+CD45 Ter119
osteogenic differentiation agenda by the tran- (called PαS) trilineage subset, although the
scription factor Ebf1/3, specializes for Cxcl12 authors of a more recent study have argued that
and SCF release and consequently for HSC main- PDGFRα+Sca-1 cells provide broader support
tenance and retention within bone marrow (Seike for CFU-F-formation and release a higher number
et al. 2018). Doubtless, niche scenery reveals a of niche mediators (like Cxcl12) than PαS cells
well-structured cell assembly with established (Zhou et al. 2014).
commitments, which reflects the singular needs Ortinau et al. (Ortinau et al. 2019) classified a
of stem cells and progenies for homing, survival, distinct periosteal stem cell population
expansion, differentiation, and egression. characterized by long-term repopulation capabil-
ity and marked by Mx1 and αSMA, which is
responsible for cortical bone regeneration and
3 Niche Maintenance repair. Notably, the Mx1+αSMA+ periosteal
and Functionality: A Matter stem cells constitute a bipolar, osteochondrogenic
of Intramural Mediators stem cell population. The authors deemed that
and Autophagic Surveillance this population has no involvement in HSC
niche dynamics, in contrast to Mx1+, Nestin-
The operational bone marrow micro-areas or, put GFP+, and LepR+ bone marrow subsets. It
more simply, niches must meet the requirements suggested that the functional schedule of the
of bone tissue, MSCs, HSCs, and progeny cells in Mx1+αSMA+ cells is controlled by specific
terms of preservation, repair, maturation, and molecular signatures and the release of mediators
peculiar skills acquisition. Soluble molecules such as the CCL5 chemokine rather than the key
and adhesive interactions between marrow HSC maintenance factor CXCL12 (Ortinau et al.
inhabitants assemble a functional architectural 2019).
complex with lineage and space-related In turn, Nestin+ and LepR+ release Cxcl12
commitments. For instance, three different MSC within the niche, which facilitates MSC homing
populations have been identified as endochondral throughout Cxcl12-Cxcr4 interaction and HSC
bone-forming progenitors, among them the prim- retention (Lai et al. 2014; Asada et al. 2017;
itive CD45 Ter119 Tie2 AlphaV+Thy 6C3 Bobis-Wozowicz et al. 2011). Additional evidence
CD105 CD200+ but also the two multipotent for the involvement of soluble factors can be seen
CD45 Ter119 Tie2 AlphaV+Thy 6C3 CD105 in the Nestin+Lepr+Sca-1+CD146+ MSCs, which
CD200 and CD45 Ter119 Tie2 AlphaV+Thy sustain LT-HSC self-renewal through the release
6C3 CD105+ progenitors (Chan et al. 2015). These of SCF and IL-11 (He et al. 2017).
distinctive mouse skeletal stem cell progenies dif- The highly dynamic bone marrow landscape is
ferentially express receptors involved in osteogene- governed by trophic molecules secreted by HSCs
sis, such as transforming growth factor (TGF)-, and MSCs, but also by mature cell types including
bone morphogenetic protein (BMP)-, and Wnt osteoblasts and hematopoietic stem cells
signaling-related receptors, which are activated by progenies. Parathyroid hormone (PTH), inter-
specific soluble factors within the niche (Chan et al. feron-γ (IFN-γ), prostaglandin F2α, bone
Autophagic Mediators in Bone Marrow Niche Homeostasis 67
morphogenetic proteins (BMPs), fibroblast growth apparatus acts as a kind of a cell homeostasis
factor 2 (FGF2) (Agas et al. 2017; Agas et al. watchdog. Indeed, the various structural gears of
2013; Marchetti et al. 2006; Sabbieti et al. 2008; this “self-cannibalism” machinery can respond to
Sabbieti et al. 2017), receptor activator of NF-κB starvation and other stressful conditions to guar-
ligand (RANKL), and osteoprotegerin (OPG) antee cell survival, but also to provide energy and
(Sharaf-Eldin et al. 2016) are some examples of raw materials for cellular turnover (Chen et al.
soluble factors involved in bone marrow and skel- 2018; Ceccariglia et al. 2020). Autophagy
etal tissue homeostasis. The secretory activity of operates in three different modes, based on the
specialized clusters prompts signaling cascades intramural executive mechanisms. In
and interactions indispensable for bone marrow microautophagy, discarded and unwanted cell
niche functionality. In this context, adrenergic products are invoked and trapped by lysosome
signals play a crucial role in delivering exceptional membrane invaginations (Gomes and Scorrano
stimuli for HSC egression, the regulation of 2013). In chaperone-mediated autophagy, cyto-
myelopoiesis and lymphopoiesis, and relief from solic chaperones recognize and trap a specific
stress signals (Maestroni 2019). pentapeptide sequence on proteins, which they
In the last decade, study of bone marrow phys- translocate and degrade into the lysosomes
iology has defined new standards and roles for (Bejarano and Cuervo 2010). Finally, in
secretomes and cellular organelles and provided macroautophagy, discarded cell components are
increasing evidence about the importance of segregated into double-membrane vesicles to
autophagy in marrow dynamics. Through its form autophagosomes. These composite
peculiar task as a recycling center, the autophagic aggregates fuse with the lysosomal membrane to
apparatus keeps under control main cellular met- give rise to the recycling core center, the
abolic features such as senescence, self-renewal, autolysosome (Agas and Sabbieti 2021;
apoptosis, and differentiation (Chen et al. 2018). Ceccariglia et al. 2020; Klionsky and Emr
Indeed, the autophagic machinery can control the 2000). Autophagosome formation is a multi-
homeostatic fate of stem cells, playing crucial stage process that requires recall of exclusive
roles in MSC and HSC expansion and their line- factors. The assembly progression begins with
age maturation agenda (Chen et al. 2018). As a the recruitment of the class III phosphatidy-
cell defense tool, autophagy responds to such linositol 3-kinase (PtdIns3K) complex (involving
stressful stimuli as inflammation, hypoxia, and beclin 1, Vps15, Vps34, Ambra1, and UVRAG)
oxidative stress, providing a stress adaption plat- and the ULK1 complex (composed of ULK1,
form for stem cell survival. Since autophagy has ATG13, ATG101, and FIP200). Ambra1 and
been correlated with HSC self-renewal and MSC TRAF6 participation further contributes to the
proliferation and differentiation, it is currently membrane maturation process (Zhang and
under evaluation as a stratagem for stem cell Baehrecke 2015). The consecutive elongation of
manipulation and related clinical applications the phagophore membrane requires the involve-
(Chen et al. 2018; Ceccariglia et al. 2020). ment of two ubiquitin-like systems. The
autophagy-related gene (Atg) family proteins
contribute, except other, to the realization of the
4 The Autophagic Apparatus ATG12–ATG5-ATG16L1 system, which
and Bone Marrow Elements: interacts with MAP 1LC3/LC3 (micro-tubule
Elective Affinities for Niche associated protein 1 light chain 3). Specifically,
Dynamics after LC3 cleavage by ATG4 into cytosol, LC3 is
converted into LC3-I and binds with phosphati-
Autophagy is the cell rejuvenation process by dylethanolamine (PE) through an ATG7/ATG3
which unnecessary material worn out organelles supportive mechanism. The LC3-I/PE interaction
and pathogens are removed from the cytoplasm with the ATG12–ATG5-ATG16L1 complex
and degraded into lysosomes. The autophagic enables the newly formed LC3-II to complete
68 D. Agas and M. G. Sabbieti
anabolic action appears to be disrupted in p62 / Osx+, and OCN+ subgroups and the increased
adult mice (Agas et al. 2020). These findings amounts of fat cells in p62 / marrow further
indicate that p62 participates as a moderator of highlight the key role of p62 in the lineage fate of
osteoblast survival and maturation. Bearing in MSCs and progenitors. This molecular hub is
mind also that p62 was primarily linked with indispensable for safeguarding niche operations
osteoclastogenesis via Nfatc1 and NF-κB signal- in terms of spatial and functional cohesion among
ing regulation (Durán et al. 2004) and controls the the MSC inhabitants (Lacava et al. 2019). More-
monocyte differentiation agenda (Zach et al. over, loss of p62 increases oncogenic stress in
2018), it is reasonable to deduce that p62 HSCs due to inefficient elimination of damaged
contributes directly to physiological bone turn- mitochondria and thus mitochondrial superoxide
over. In fact, intramuscular administration of accumulation within the niche (Nguyen et al.
p62 DNA in ovariectomized adult (5 m) mice 2019).
was able to invert the osteoporotic/inflammatory In sum, our information on the autophagy-
bone profile. This was due to augmented expres- related and unrelated features of p62 and their
sion of osteo-inductive markers with simulta- influence on bone marrow dynamics shows that
neous anti-inflammatory action. Exogenous this protein serves as an indispensable mechanis-
delivery of p62 was able to calm tic gear for the operational activities of MSCs and
pro-inflammatory cytokines and chemokines HSCs (Fig. 2). Since p62 controls the differentia-
such as TNF-α, IL-6, IL-1β, and IL-17, and in tion fate and sub-population expansion of MSCs
parallel it moderated osteoclastogenic promoters as well as the homing, maintenance, and myeloid
such RANKL and NF-/κB within the bone mar- lineage maturation of HSCs (monocytes and
row (Sabbieti et al. 2015). osteoclasts), it is seen as a jack of all trades not
There are conflicting views of the role of ana- only in steady state but also in pathologies. This
bolic p62 on monocyte/osteoclast maturation. receptor carries out a wide range of functional
Some studies report that p62 mutations improve duties affecting bone marrow cell behavior as
osteoclast nucleation and increase their size with well as inflammatory and oxidative tissue
concomitant reduction of bone volume defense, the regulation of survival intramural
(Daroszewska et al. 2011; Hiruma et al. 2008; signals and the accomplishment of autophagy.
Kurihara et al. 2007). Along this line, the absence
of p62 has been related with mature osteoclast
expansion in vitro. A solidly grounded interpreta- 6 The Role of Atg7
tion of these results suggests that p62 can exert a (Autophagy-Related Protein 7)
regulatory function on naïve monocytes, directing in Bone Marrow Dynamics
them toward osteoclast lineage commitment
(Zach et al. 2018). Macroautophagic apparatus assembly is a com-
Data obtained from p62 / aged mice plex, highly coordinated process in which two
(1y and 2y old) showed augmented bone marrow main mechanistic units organize a series of func-
adipogenesis with concurrent reduction of MSC tional gears. The over 30 Atg proteins identified
and pre /mature osteoblast populations. It is well to date contribute to these two ubiquitin-like con-
established that increased bone marrow fat can jugation systems. Atg1, Atg9, phosphatidyl-
lead to HSC dysregulation and MSC regression inositol 3-kinase complex, and microtubule-
(Lacava et al. 2019; Adler et al. 2014). In this associated Protein 1 Light Chain 3 congregate in
context, enhanced marrow adiposity observed in the LC3 system, while Atg5, Atg10, Atg12, and
the absence of p62 causes the accumulation of Atg16 give rise to the Atg12 system (Feng et al.
reactive oxygen species (ROS) and hemosiderin 2015). Atg7 carries out a special enzymatic func-
and consequently the formation of a toxic habitat tion in both systems, indispensable for the con-
for the homing of MSCs and HSCs (Lacava et al. gregation and expansion of autophagosomal
2019). The decline of the mesenchymal Nestin+, membranes (Feng et al. 2015; Xiong 2015).
70 D. Agas and M. G. Sabbieti
Fig. 2 p62 within an autophagy direct or indirect action orchestrates a wide range of bone marrow homeostatic features
such as osteoblastogenesis, osteoclast maturation, and HSC regulation
The results of recent research have added more Vav-Atg7 / mice culminated with HSC
evidence linking Atg7 with the dynamics of bone multilineage cytopenia and gradual bone marrow
marrow stem and mature cells. In fact, Atg7 has failure (Mortensen et al. 2011a). Recent findings
been correlated with HSC maintenance and support the view that Atg7 in an autophagy-
multilineage maturation, as well as MSC survival. related and autophagy-unrelated fashion is able
In addition, Atg7 is thought to have differentiated to oppose hematopoietic aging. Specifically,
commitments in bone cells. Mice with Atg7 con- Atg7 / mice showed disrupted autophagy,
ditional deletion in HSCs and hematopoietic which leads to hematopoietic growth retardation
progenitors (Vav-Atg7 / mice) revealed and reduced life cycle (Fang et al. 2019). In
impaired HSC activity and maintenance, with addition, vav-Atg7 / mice displayed impaired
consequent restricted myeloid and erythroid pro- monocyte differentiation into macrophages, an
genitor pool. The absence of Atg7 within the indication that Atg7-related autophagy supports
Lin Sca-1+c-Kit+ (LSK) cell subset incites the the morphological and operational maturation of
intracellular amassing of high membrane poten- macrophages induced by colony-stimulating fac-
tial mitochondria, augments mitochondrial super- tor-1(Csf-1) (Jacquel et al. 2012).
oxide release, and increases DNA damage In an autophagy-unrelated fashion, Atg7
(Mortensen et al. 2011a). Impaired autophagy in exerts a crucial role in nucleosome/chromatin
HSCs/progenitors induced by Atg7 loss leads to assembly, a process directly correlated with the
drastic reduction of the LSK pool and potential aging process of the myeloid CD11b+Ly6G
development of malignant myeloproliferative dis- bone marrow population. Atg7 deletion signifi-
order in mice. The adverse effects observed in cantly reduces the number of CD11b+ myeloid
Autophagic Mediators in Bone Marrow Niche Homeostasis 71
cells and confers an aging phenotype on this homeostasis. In fact, Dmp1-Cre; Atg7-f/f mice
blood lineage subgroup (Fang et al. 2020). displayed disrupted bone turnover with altered
Since niche cohesion requires physiological osteoclast and osteoblast population, which in
cross-talk among inhabitants, it is understandable turn resulted in inflammaging-like osteopenia
that a pathological hematopoietic phenotype is (Onal et al. 2013).
seen in diminished MSC pool and bone mineral To summarize, the vast majority of Atg7 loss-
density with concurrent increase of oxidative of-function studies underlined the crucial tasks of
stress and accelerated cell aging. The importance this archetypal autophagic mediator in MSC and
of autophagy in MSC and progenitor descendants HSC maintenance and lineage commitment and
has been studied extensively, albeit with contra- in bone tissue rejuvenation (Fig. 3). Undoubtedly,
dictory results in some cases. Atg7 abrogation by Atg7 within an autophagy-related mode, but also
short hairpin RNA in irradiated human MSCs as distinct molecular hub, plays a remarkable role
pretreated with starvation caused increased ROS in bone and bone marrow dynamics. Further stud-
levels and DNA damage. These outcomes under- ies are required to better elucidate the autophagy-
line the key role of Atg7-related autophagy in unrelated accomplishment of Atg7 in bone mar-
safeguarding MSC integrity and stemness (Hou row architecture.
et al. 2013).
In a different view, Atg7 knockdown in MSCs
exposed to critical conditions (such as hypoxia, 7 Conclusions
serum deprivation, and oxidative stress)
safeguards the cells against stressful micro- The bone marrow is the extraordinary, highly
environmental stimuli. These outcomes indicate compartmentalized and tremendously inter-
that as a functional autophagic gear, Atg7 plays a connected multicellular core of the bones. Its oper-
cytoprotective role in a physiological or a mild ational micro-areas ensure MSC and HSC homing,
pathological milieu, while in severe MSC stress expansion, and lineage commitment, as well as the
settings, autophagy leads to cell death (Molaei egression and in-house allocation of progenitor and
et al. 2015). mature cells through the release and physical inter-
Autophagy is a well-documented moderator action of cytokines, chemokines, growth factors,
for MSC differentiation toward osteoblasts as and other molecules. For clinical applications such
well as chondrocytes. In a recent work reporting as bone marrow transplants, treatment of
the contribution of Atg7-related autophagy to hematopoietic diseases, and the use of MSCs for
chondrogenesis, the authors argued that Atg7 is their anti-inflammatory and regenerative
an indispensable ring for the transition of mesen- characteristics, it is important to understand the
chymal stem cells and pre-chondrocytes to behavior of the pluripotent stem cells and the
mature cells and that its absence causes endoplas- differentiated bone marrow cell inhabitants. In the
mic reticulum stress-mediated chondrocyte death last two decades, research efforts have revealed the
(Kang et al. 2017). Moreover, in Atg7flox/flox essential role of autophagy in the maintenance,
mice, the impaired autophagy in chondrocytes self-renewal, and maturation of MSCs and HSCs.
triggered severe growth retardation of the tibia They have also blazed new paths for bone and bone
and femur, followed by mechanistic disorders in marrow investigation by clarifying the importance
chondrocytes (Horigome et al. 2020). Parallel of the main autophagic mediators, such as p62 and
research revealed that conditional ablation of the members of the Atg family, and reporting on
Atg7 or Atg5 in murine chondrocytes aggravated related gain- and loss-of-function outcomes. These
axial and appendicular skeletal growth due to archetypal autophagic gears carry out a variety of
caspase-dependent apoptosis of these cells tasks, some strictly related with the autophagy
(Vuppalapati et al. 2015). Likewise, suppression complex and others unrelated, as individual
of Atg7-dependent autophagy in murine mediators partaking in homeostatic signaling
osteocytes significantly changes bone tissue cascades. For instance, the presence of p62 in
72 D. Agas and M. G. Sabbieti
Fig. 3 Atg7 autophagy-related and autophagy-unrelated mode of action stimulates bone turnover and regulates MSC
and HSC homeostatic features
MSCs has been linked to osteoblastogenesis; simi- Agas D, Sabbieti MG (2021) Archetypal autophagic
larly, the absence of Atg7 has been associated with players through new lenses for bone marrow stem/
mature cells regulation. J Cell Physiol 236:6101–6114
deleterious effects on bone and bone marrow Agas D, Marchetti L, Capitani M, Sabbieti MG (2013) The
homeostasis. Ongoing studies seek to deepen our dual face of parathyroid hormone and prostaglandins in
knowledge of the actions of autophagic markers the osteoimmune system. Am J Phys Endocrinol
within the labyrinthine bone marrow interface. Metab 305:E1185–E1194
Agas D, Marchetti L, Douni E, Sabbieti MG (2015) The
unbearable lightness of bone marrow homeostasis.
Cytokine Growth Factor Rev 26:347–359
Agas D, Gusmão Silva G, Laus F, Marchegiani A,
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# Springer Nature Switzerland AG 2021
Published online: 2 November 2021
77
78 J. Hatina et al.
(Sox17+ Sox2) and embryonal carcinoma members markedly impact the final regulatory
(Sox17 Sox2+). Strikingly, both factors complex outcome, as exemplified in TGCT above. A simi-
with Oct4 to cooperatively bind composite regu- lar complexity may be pursued within the Klf
latory elements mentioned above, with a subtle family (Kim et al. 2017a). If not carefully
preference for one or another particular version of checked for these complex expression patterns,
them. The Sox17-containing complexes prefer the interpretation of a mere finding of
binding to a particular form called compressed overexpression of pluripotency factor(s) in cancer
composite element, lacking a single G between might be rather difficult (see Table 1).
the respective Sox- and Oct4-binding motifs,
whereas Sox2 complexes prefer canonical com-
posite elements involving the spacer guanosine 3.1 Pluripotency Transcription
residue. Consequently, downstream genes Factors in Cancer Stemness
activated by either Sox-Oct4 complexes differ in
part, with seminoma specific expression of There is ample evidence that at least a good part
oncogenes IGF-1 and c-myc and a broader spec- of tumours keeps intrinsic hierarchy analogical to
trum of pluripotency network genes in embryonal that found in continuously renewing tissues or
carcinoma (Jostes et al. 2020). even at the beginning of development. Cancer
There is a plethora of studies available in the stem cells (CSCs) have been operationally
scientific literature exploring expression of defined as a cancer cell population endowed
pluripotency transcription factors (both individu- with long-term proliferative capacity due to their
ally and in combinations) in a wide spectrum of self-renewal and self-protection, thus driving con-
cancer types. It should be noticed, nevertheless, tinuous tumour growth in primary outgrowth or at
that such findings can be confounded by the recurrence or on metastatic spread. The self-
existence of multiple splice variants and protection mechanisms can be exploited to
pseudogenes, which have been identified for approach these cells experimentally, with side
Oct4, Nanog and Sall4 (Bernhardt et al. 2012). population and Aldefluor assays developing into
As for the former, only one isoform, Oct4A, almost universal methods of their purification,
participates in the pluripotency regulatory net- reflecting their high xenobiotic efflux pump
work. Two other splice variants, known as activity (especially ABCB1 and ABCG2) and
Oct4B and Oct4B1, may be overexpressed in xenobiotic detoxification activity (especially
cancer, with functional implications quite differ- aldehyde dehydrogenases ALDH1A1 and
ent from conferring pluripotency stemness. In ALDH1A3), respectively. Alternatively, a series
contrast, Sall4 is expressed in two splice variants, of cell surface markers, sometimes reminiscent of
Sall4A and Sall4B, with the B isoforms being niche organization of the respective tissue of
significantly more proficient in promoting both origin, can be used as well, including CD133,
pluripotency (Rao et al. 2010) and cancer CD44, CD49f, CD90 and a few more in specific
(Ma et al. 2006). In addition, pluripotency tran- cancer types (e.g. haematopoietic stem cell
scription factors feature numerous pseudogenes markers in leukaemias). While the self-
(6–8 for Oct4, 10 for Nanog and up to 16 for protection-based approaches reflect an intrinsic
Stella) (Bernhardt et al. 2012; Pain et al. 2005), biological property of cancer stemness, the cell
and some of them might be expressed, again with surface markers may frequently lack any func-
a very specific function distinct from their role in tional link. They are often combined, both with
pluripotent stem cells. As for the Sox2, it should each other and with the self-protection-based
not be forgotten that it is a member of a particu- assays. Functionally, cancer stem cells feature a
larly complex gene and protein family counting at series of traits, like high clonogenic activity, espe-
least 20 members (Li et al. 2016), with closely cially in anchorage-independent settings (either in
related DNA-binding specificities, and the semisolid media like agar, agarose or methylcel-
biological relationships between individual lulose, or in suspension as cancer spheres), and,
Table 1 Expression of pluripotency transcription factors in cancer
82
SOX2
Cancer type Increased expression Prognosis/stage/clinical outcome Decreased expression/no Prognosis/stage/
expression/loss of expression clinical outcome
Breast Ben-Porath et al. (2008); Chen et al. (2008) Poor prognosis (Chen et al. 2008)
Colorectal Saiki et al. (2009) Lymph node metastasis (Saiki et al. 2009)
Oesophageal Wang et al. (2009) Poor survival (Wang et al. 2009)
squamous
Gastric Tian et al. (2012); Chen et al. (2016) Enhanced tumorigenicity (Tian et al. 2012) Otsubo et al. (2008); Wang et al. Poor prognosis
Better treatment outcome (Chen et al. (2015) (Otsubo et al. 2008)
2016) Worse clinical
outcome (Wang et al.
2015)
Glioma Guo et al. (2011) High tumour stage (Guo et al. 2011)
Head and neck Lee et al. (2014) Tumour recurrence and poor prognosis Bayo et al. (2015) Worse survival (Bayo
squamous cell (Lee et al. 2014) et al. 2015)
Melanoma Hadjimichael et al. (2015)
Lung Li et al. (2013a) Enhanced tumorigenicity (Chen et al.
Chen et al. (2012a, p. 2) 2012a)
Wilbertz et al. (2011); Toschi et al. (2014) Better prognosis in early stage (NSCLC)
Better prognosis (Wilbertz et al. 2011)
Ovarian Pham et al. (2013); Robinson et al. (2021) A role in tumour relapse (Robinson et al.
2021)
High-grade tumour (Pham et al. 2013)
Better overall survival (Belotte et al. 2015)
Oral squamous Fu et al. (2016); Ghazi et al. (2020) Earlier stage (Fu et al. 2016)
High grade (Ghazi et al. 2020)
Nanog
Cancer type Increased expression Prognosis/stage/clinical outcome Decreased expression/no Prognosis/stage/
expression/loss of expression clinical outcome
Breast Ben-Porath et al. (2008); Lu et al. (2014) Advanced stage (Lu et al. 2014)
Colorectal Saiki et al. (2009) No clinical significance (Saiki et al. 2009)
Meng et al. (2010) Poor prognosis and lymph metastasis
(Meng et al. 2010)
Gastric Lin et al. (2012) Advanced stage (Lin et al. 2012)
Gliomas Guo et al. (2011) High tumour stage (Guo et al. 2011)
J. Hatina et al.
Lung Chiou et al. (2010); Li et al. (2013b); Sławek High grade/poor prognosis (Sławek et al.
et al. (2016) 2016)
Oral squamous Chiou et al. (2008); Belotte et al. (2015); Fu Advanced stage (Fu et al. 2016;
et al. (2016); Vijayakumar et al. (2020) Vijayakumar et al. 2020)
Ovarian Lee et al. (2012); Amsterdam et al. (2013); Worse prognosis (Siu et al. 2013)
Siu et al. (2013)
Prostate Jeter et al. (2011) Worse prognosis (Jeter et al. 2011)
Oct4
Cancer type Increased expression Prognosis/stage/clinical outcome Decreased expression/no Prognosis/stage/
expression/loss of expression clinical outcome
Bladder Xu et al. (2007); Hatefi et al. (2012) Poor prognosis (Xu et al. 2007; Hatefi et al.
Uhlén et al. (2015) 2012)
Favourable prognostic factor (Uhlén et al.
2015)
Breast Ben-Porath et al. (2008); Shen et al. (2014); Supressed the metastatic potential (Shen
Soheili et al. (2017) et al. 2014)
Colorectal Saiki et al. (2009) No clinical significance (Saiki et al. 2009)
Oesophageal Wang et al. (2009) Poor survival (Wang et al. 2009)
squamous
Gastric Matsuoka et al. (2012) Good prognosis (Matsuoka et al. 2012)
Pluripotency Stemness and Cancer: More Questions than Answers
Gliomas Guo et al. (2011) High tumour stage (Guo et al. 2011)
Head and neck Koo et al. (2015) Advanced stage (Koo et al. 2015)
squamous
carcinoma
Intrahepatic Zhang et al. (2019) Aggressive tumour and poor prognosis
cholangiocarcinoma (Zhang et al. 2019)
Lung Chiou et al. (2010); Chen et al. (2012b); Li High grade/poor prognosis (Li et al., 2012) Moreira et al. (2010)
et al. (2013b)
Medulloblastoma Rodini et al. (2012) Poor survival (Rodini et al. 2012)
Oral squamous Chiou et al. (2008); Fu et al. (2016); Advanced stage (Ghazi et al. 2020)
Vijayakumar et al. (2020) Non-significant role in regulation of
tumour behaviour (Vijayakumar et al.
2020)
Prostate Matsuoka et al. (2012) Good prognosis (Matsuoka et al. 2012)
(continued)
83
Table 1 (continued)
84
c-Myc
Cancer type Increased expression Prognosis/stage/clinical outcome Decreased expression/no Prognosis/stage/
expression/loss of expression clinical outcome
Breast Ben-Porath et al. (2008)
Colorectal Saiki et al. (2009) No clinical significance (Saiki et al. 2009)
Glioblastoma Zheng et al. (2008) Advanced stage
Glioma Wang et al. (2008)
Hepatocellular Wang et al. (2002b) Poor prognosis (Wang et al. 2002b)
Lung Sławek et al. (2016) Worse survival (Johnson et al. 1996)
Ovarian Uhlén et al. (2015) Unfavourable prognostic factor (Uhlén
et al. 2015)
Renal Uhlén et al. (2015) Unfavourable prognostic factor (Uhlén
et al. 2015)
Urothelial Uhlén et al. (2015) Unfavourable prognostic factor (Uhlén
et al. 2015)
Uterine cervix Riou et al. (1987) Worse relapse-free survival rate (Riou et al.
1987)
Klf4
Cancer type Increased expression Prognosis/stage/clinical outcome Decreased expression/no Prognosis/stage/
expression/loss of expression clinical outcome
Breast Yu et al. (2011); Okuda et al. (2013) Advanced tumour
Pandya et al. (2004)
Colon Patel et al. (2010) Better prognosis
(Patel et al. 2010)
Head and neck Tai et al. (2011) Poor prognosis Wang et al. (2002a)
squamous cell
Lung Hu et al. (2009); Zhou et al. Aggressive tumour
(2010, p. 4); Gómez et al. (2014) (Gómez et al. 2014)
Renal Uhlén et al. (2015) Favourable prognostic marker (Uhlén et al.
2015)
Thyroid Wang et al. (2019)
J. Hatina et al.
Sall4
Cancer type Increased expression Prognosis/stage/clinical outcome Decreased expression/no Prognosis/stage/
expression/loss of expression clinical outcome
Cholangiocarcinoma Deng et al. (2015) Worse prognosis (Deng et al. 2015)
Oesophageal Forghanifard et al. (2014), He et al. (2016) Tumour invasion and metastasis
squamous (Forghanifard et al. 2014)
Endometrial Li et al. (2015) Worse survival (Li et al. 2015)
Glioma Zhang et al. (2015) Poor prognosis (Zhang et al. 2015)
Leukaemia Yang (2018) Disease progression (Yang 2018)
Liver Oikawa et al. (2013); Yong et al. (2013) Tumour growth and resistance to 5-FU
(Oikawa et al. 2013)
Aggressive phenotype (Yong et al. 2013)
Hepatoblastoma Zhou et al. (2016) Worse overall survival (Zhou et al. 2016)
Hepatocellular Yong et al. (2013); Liu et al. (2014, p. 4); Poor prognosis (Liu et al. 2014; Jung et al.
Jung et al. (2016) 2016)
Aggressive phenotype (Yong et al. 2013)
Myelodysplastic Wang et al. (2013) Worse survival rate (Wang et al. 2013)
syndromes
Ovarian Yang et al. (2016) Advanced stage (Yang et al. 2016)
Renal Che et al. (2020) Worse survival (Che et al. 2020)
Pluripotency Stemness and Cancer: More Questions than Answers
where appropriate, high tumorigenic activity after cisplatin chemosensitivity, might directly derive
(xeno)transplantation into a suitable host. Inher- from the biological activity of pluripotency tran-
ent to the existence of self-protection mechanisms scription factors, especially Oct4 (Gutekunst et al.
is their intrinsic therapeutic resistance, both as an 2013). Importantly, during the development of
experimental approach to characterize them and the chemoresistant disease, tumour cells eliminate
within clinical context, thus underlying clinical expression of both Oct4 and Nanog (Taylor-
disease therapy resistance and relapse (Hatina Weiner et al. 2016) and highly probably switch
et al. 2013). their stemness mechanism to another biological
The manifestation of pluripotency stemness, basis.
especially expression of the core pluripotency In this way, the pluripotency stemness (espe-
transcription factors, has been invoked as another cially Oct4-dominated) might be rather antitheti-
unifying characteristic of cancer stemness. In cal to cancer stem cell self-protection. Indeed,
many cases, nevertheless, this is based more on when analysed in detail, Oct4 seems not to be a
an a priori assumption rather than a hypothesis- crucial factor responsible for the cancer stemness
free evidence, by just demonstrating that cancer (Menendez et al. 2020; Robinson et al. 2021), and
cell populations enriched in cancer stem cells by within the context of the pluripotency network,
any method discussed above express higher level Sox2 might dominate instead these cancer stem
of pluripotency transcription factors or their cell populations. It may be interesting to note in
mRNAs than the corresponding non-stem this context that Sox2 can participate in additional
fractions, and it is plagued by many of the intrin- signalling pathways, for example, Hippo-Yap
sic weaknesses discussed above, including the (Basu-Roy et al. 2015) or Hedgehog (Justilien
existence of pseudogenes, isoforms and closely et al. 2014). On the other hand, the impact of
related family members, which are rarely taken Sox2 is highly context-dependent. While
into account. On the other hand, it now seems to characterized as an amplified oncogene in
be really experimentally substantiated that a part oesophageal and lung squamous cell carcinoma,
of cancer cases (either clinical tumours or cancer a transgenic model revealed its tumour-
cell lines) use core pluripotency transcription suppressive role within the context of gastric car-
factors to foster their stem-like populations. The cinogenesis, principally due to its interference
most revealing evidence has been achieved by with the Wnt-β-catenin signalling (Sarkar et al.
using reporter constructs driven by the 2016), and even within the context of the former
multimerized Sox-Oct4 composite regulatory group of tumours, its high expression is
elements, either originating from the Sox2 gene associated with a favourable prognosis (Wilbertz
itself (SRR2 enhancer – Iglesias et al. 2014; Wu et al. 2011). A similar tumour type- and context-
et al. 2012) or from the Nanog gene (SORE6 specific oncogenic or tumour-suppressive role has
enhancer – Keysar et al. 2017; Menendez et al. been evidenced for the Klf4 as well (Ray 2016).
2020; Pádua et al. 2020; Tang et al. 2015), which Plausibly, this context dependence might be at the
turned out to be very useful across different can- onset of discrepancies with the earlier report
cer types, but usually just in a fraction of tumour showing that activation of pluripotency stemness
samples. This implies that pluripotency stemness circuit is associated with high-grade poor progno-
might be just one of multiple possible stemness sis tumours (Ben-Porath et al. 2008).
mechanisms, complementing other mechanisms
within or among individual tumours, within the
context of the concept of cancer stem cell hetero- 4 Induced Pluripotent Stem Cells
geneity, reviewed extensively in this series and Induced Pluripotent
(Birbrair 2019a, b). This situation is best Cancer Cells
illustrated in TGCT that can be taken as a proto-
typic tumour type driven by the pluripotency Perhaps the most conclusive evidence for a causal
stemness circuitry. Indeed, specific clinical role of the pluripotency transcription factors in
characteristics of this tumour, like extreme pluripotency stemness lies in their ability to
Pluripotency Stemness and Cancer: More Questions than Answers 87
et al. 2010), lung carcinoma cells (Mahalingam Yamanaka factors into the bulk culture, it pro-
et al. 2012) as well as a spectrum of various moted reprogramming. This promoting effect was
sarcomas (Zhang et al. 2013). This way of cancer strictly non-cell-autonomous, i.e. based on a para-
reprogramming has even been suggested as a crine activity of “loser” cells towards the success-
possible way to a future therapeutic strategy fully reprogrammed cells. If tested for cell-
(Gong et al. 2019; Miyoshi et al. 2021; Yilmazer autonomous effect, i.e. in individual cells that
et al. 2015). received both activated H-ras and the polycis-
tronic vector coding for Yamanaka factors, then
activated H-ras behaved dramatically inhibitory
4.1 Why? (Ferreirós et al. 2019). Absolutely the same has
been described for another crucial cancer onco-
In order to tackle this paradox, we have to con- gene, Yap, the downstream effector of the Hippo
centrate on biological differences between pathway (Hartman et al. 2020). Of note, such a
pluripotency reprogramming (or pluripotency distinction between cell-autonomous and non-
stemness) and tumorigenic transformation. cell-autonomous effects is difficult to get unless
Indeed, besides the parallels between the two deliberately pursued, thus explaining a lot of
processes, there are signalling pathways that are seemingly contradictory reports (Chung et al.
frankly antithetical, and perhaps the most impor- 2016; Qin et al. 2012; Tamm et al. 2011).
tant among them is the MAPK pathway. We have Importantly, both MAPK–AP-1 and Hippo-
already mentioned that inclusion of the specific Yap signalling pathways have dramatically dif-
MEK inhibitor PD0325901 was an important step ferent impacts in both normal adult stem cells and
in formulating improved ESC culture media to cancer stem cells. Jun combines with
prevent the FGF-4-driven autodifferentiation Wnt-β-catenin signalling in an intricate regulatory
activity. The downstream transcription factors circuit governing ISCs and colorectal cancer stem
activated by the MAPK pathway, especially the cells and operating both feedforward (Nateri et al.
transcription factors of the AP-1 family, 2005; Sancho et al. 2009) and feedback (Kabiri
consisting of various Jun and Fos dimers, have et al. 2018; Harmston et al. 2021) pathways. Side
been convincingly characterized as one of the population cells of various sarcomas, neuroblas-
most pronounced inhibitors of both pluripotency toma and urothelial carcinoma are dependent on
stemness and pluripotency reprogramming (Liu active MAPK signalling; strikingly, in these
et al. 2015). Along the same line, pharmacologic cancers active MAPK signalling is mechanisti-
inhibition of activated B-Raf was reported to cally linked to the expression of pluripotency
increase the pluripotency reprogramming of mel- transcription factors (Tsuchida et al. 2008;
anoma cells (Castro-Pérez et al. 2019), and Hepburn et al. 2012). In addition, Jun-Fos
activated H-ras was shown to markedly decrease constitutes a central regulatory hub of Ewing
iPSC reprogramming efficiency. This last exam- sarcoma side population cells (Hotfilder et al.
ple revealed a hitherto unprecedented cellular 2018), and Jun is the major effector of the
complexity of the reprogramming process, never- stemness-promoting effect of the connective tissue
theless. The pluripotency reprogramming process growth factor (CTGF) in head and neck squamous
is inherently very inefficient, with success rate of cell carcinoma (HNSCC) (Chang et al. 2013).
the original Yamanaka protocol far below 1%, Intriguingly, in ESCs and iPSCs, Jun-imposed
and there are complex signalling interactions transcriptome included downregulated pluripotency
between the successfully reprogrammed cells stemness genes including Nanog, Sall4, Oct4, Sox2
and the vast majority of cells that fail to achieve and Esrrb, and it was almost completely opposite to
pluripotency stemness – the latter can be seen as a the Oct4-induced transcriptome (and, along the
sort of niche cells promoting the acquisition of same lines, transcriptomes imposed by Oct4 and
stemness by the minority of “winners”. If endogenous Jun inhibitors, either a Jun-dominant
activated H-ras was transduced together with negative mutant or Jun dimerization protein 2,
Pluripotency Stemness and Cancer: More Questions than Answers 89
were practically identical (Liu et al. 2015)). In con- involving several molecularly discernible
trast, MEK inhibitor-treated sarcoma and urothelial subpopulations (Hatina et al. 2019).
carcinoma cells significantly reduced their SP frac- The existence of cancer (stemness)-promoting
tion and tumorigenicity (Tsuchida et al. 2008; signalling pathways that at the same time strongly
Hepburn et al. 2012) instead of activating stemness inhibit, in cell-autonomous manner, the
as seen in ESCs and iPSCs. In CTGF-stimulated pluripotency reprogramming could provide a
HNSCC cells, Jun is rapidly activated and subse- straightforward explanation for the loss of
quently directly activates pluripotency core factors tumorigenicity observed in iPCCs. In addition,
Oct4, Sox2 and Nanog. Even this last aspect turned it has been repeatedly reported that efficiency
out to be more complicated if followed at single-cell of pluripotency reprogramming is inversely
resolution level. Based on the results described correlated to genomic complexity of cancer
above, we applied CTGF to aggressive sarcoma cells. For example, the reprogramming efficiency
cells, and we indeed observed a dramatic effect on gradually decreased along the melanoma progres-
anchorage-independent clonogenicity. Immunoflu- sion (Castro-Pérez et al. 2019), and genomically
orescence staining failed to evidence any Oct4 simpler tumours like hepatoblastoma were much
induction, and staining for Sox2 and Jun revealed easier to reprogram than the corresponding com-
heterogeneous and only partially overlapping pat- plex cancer like hepatocellular carcinoma (Kuo
tern (Fig. 1). Plausibly, cancer stem cells should be et al. 2016). High mutational load of tumour cells
understood more as a collective term to describe and their high heterogeneity even within individ-
cells bearing special functional characteristics and ual tumours or individual cancer cell lines,
Fig. 1 The murine fibrosarcoma cell line JUN3 (Hatina 1:400 and anti-SOX2 rabbit polyclonal antibody at 1:200
et al. 2003) was routinely cultured in high glucose dilution) followed by extensive washing and 2 h incuba-
(4500 mg/l) Dulbecco’s modified Eagle’s medium tion with secondary antibodies (rabbit anti-mouse Atto
supplemented with 10% foetal calf serum, penicillin 488; green fluorescence and goat anti-rabbit – TRITC;
(final concentration 100 U/ml) and streptomycin (final red fluorescence, 1:400 dilution, room temperature),
concentration 100 μg/ml) at 37 C in a humidified atmo- extensively washed and mounted. (A) Colonies in methyl-
sphere containing 5% CO2. The cells were treated with the cellulose were counted after 10 days; pictures were taken
CTGF (10 ng/ml) after 24 h serum starvation for 5 days by the Olympus IX 81 inverted microscope equipped with
and processed for anchorage-independent clonogenicity the Hamamatsu Orca-ER camera. Bar: 200 μm. (B) The
assay by suspending 10,000 cells in 15% methylcellulose statistical significance is based on permutational t-test
[109] or fixed in 4% formaldehyde in PBS for 30 min, (p ¼ 0.004). Each point represents an individual well.
blocked in 1% BSA for 1 h and processed for immunoflu- (C) Immunofluorescence pictures were taken by the Olym-
orescence with overnight incubation at 4 C with primary pus IX 81 inverted microscope equipped with the
antibodies (anti-c-JUN mouse monoclonal antibody at Hamamatsu Orca-ER camera. Bar: 100 μm
90 J. Hatina et al.
undergo reprogramming and achieve BMP4 (secreted in doses of about 500 pg/ml
pluripotency, is not yet clear. detected in conditioned media) is protumorigenic
Parallels and differences in epigenetic pro- within the context of colorectal carcinoma, includ-
cesses taking place during iPSC and iPCC ing the promotion of anchorage-independent
reprogramming can have two additional clonogenicity (one of the typical stem cell
consequences. First, quite analogical to the characteristics) (Yokoyama et al. 2017), whereas
EMT-MET plasticity observed during normal high-dosed BMP4 (100 ng/ml) has been repeatedly
reprogramming, premature termination in cancer identified as a CSC differentiation factor, both for
cell reprogramming may accentuate the invasive- colorectal carcinoma (Lombardo et al. 2011) and
ness of cancer cells, which can be of benefit for glioblastoma, being even considered as a promising
identification of novel invasion-associated cancer therapy for the latter (Nayak et al. 2020). Moreover,
genes (Knappe et al. 2016). Second, unlike nor- the iPSC reprogramming efficiency, as well as the
mal pluripotency reprogramming, the EMT-MET quality (i.e. the ability to develop into an intact
succession may not be finished during the cancer mouse organism), also crucially depends on the
pluripotency reprogramming, yielding a metasta- respective ratio of reprogramming factors, espe-
ble EMT-MET transition state (Hiew et al. 2018), cially Oct4 and Sox2, with Oct4 (high) and Sox2
which in fact may be quite typical for metastasis- (low) combination being one to two orders of mag-
competent cancer cells (Celià-Terrassa and Kang nitude more efficient than other combinations
2016) and hence may provide a valuable insight (Nagamatsu et al. 2012), and this combination was
into the biology of cancer metastasis. also characteristic of the reprogramming mixture
yielding iPSCs that could develop into “all-iPSCs”
embryos (i.e. derived entirely from iPSCs) (Carey
5 Dose Dependency et al. 2011). Importantly most tumours, unlike nor-
of Pluripotency Factors mal differentiated cells, express pluripotency factors
(especially Sox2 – see above), which adds to the
Another point deserving a brief discussion is that transfected reprogramming factors and in the end
the pluripotency factors feature a pronounced dose possibly profoundly influences the
dependency of action. In particular, they act as cancer reprogramming, both regarding the
stemness factors only within quite a narrow range, reprogramming efficiency and the phenotype of
and underexpression, but also overexpression trig- the resulting iPCCs. Adding to the complexity,
ger differentiation. This has been reported for both there are “close relatives” of both Sox2 and Klf4
Oct4 (Niwa et al. 2000) and Sox2 (Kopp et al. within the respective protein families, which can
2008); interestingly with regard to the latter, the partially substitute for them during the pluripotency
impact on transcription of downstream target reprogramming (Jiang et al. 2008), and for which a
pluripotency genes dramatically changes along protumorigenic role and corresponding
with elevating the Sox2 level, switching from acti- overexpression have been described in cancer –
vator as expected for the functionality of the this is, for example, the case of Sox3 (Guo et al.
pluripotency network to transcriptional repressor 2018; Marjanovic Vicentic et al. 2019) or Klf5 (Liu
(Boer et al. 2007). The same dose dependency is et al. 2020; Siraj et al. 2020).
true for the BMP4 as a crucial stemness signalling
molecule as well. It was consistently observed that
low to very low dosed BMP4 (10 ng/ml for ESCs, 6 Conclusion
0.01–0.1 ng/ml for MSCs) acts as a powerful
stemness factor (Vicente López et al. 2011), The pluripotency reprogramming has been
whereas doses of 50 ng/ml to 100 ng/ml are consis- mostly conceived within the context of regenera-
tently used to induce differentiation (Cordonnier tive medicine, to provide in theory unlimited
et al. 2011). Moreover, this dose-dependent effect individual patient-matched source of stem cells
seems to be well conserved in cancer – autocrine to be differentiated in tailored fashion and
92 J. Hatina et al.
transplanted back in order to replace disease- the context of personalized medicine (Papapetrou
damaged cells. Soon it was realized that in addi- 2016) seems to be a very remote vision at best. Of
tion to this possible direct clinical application, course, a seemingly more straightforward solu-
iPSCs offer tremendous possibilities for experi- tion appeared to be to start the reprogramming
mental modelling of numerous human diseases, process with very tumour cells, which would fix
including various neurodegenerative diseases like all the complex (and in part still elusive) cancer
Huntington, Alzheimer and Parkinson diseases, genetic abnormalities in a series of tumour-
amyotrophic lateral sclerosis as well as cardiomy- derived iPSCs (or better in current nomenclature
opathy, liver metabolic disorders and urinary tract iPCCs). In our view, elaborated above, this strat-
and prostate disorders (Argentati et al. 2020). egy is very problematic as well. Experience
Cancer couldn’t be expected to stand aside, but shows that genetic complexity is antithetical to
it increasingly turns out that modelling cancer reprogramming capacity and a lot of signalling
with the help of iPSC methodology will be rather pathways have contradictory effects in cancer
a daunting task. Cancer is not a single disease, but cells and in pluripotent cells. Pluripotency
a group of many hundreds of individual reprogramming in highly complex advanced
diagnoses, with unifying basic biology but each tumours would thus catch at best a fraction of
with its own genetic and epigenetic landscape. cancer cells, and the most aggressive cells
While modelling some genetically simpler cancer would be probably missed. Another theoretical
phenotypes like certain leukaemias (Turhan et al. concept might be that tumour-derived iPSCs
2019) or very initial stages of hereditary (Zhu would be enriched in cancer stem cells, the most
et al. 2018) and in near perspective hopefully therapy-resistant functional cancer cell subset;
also sporadic cancers might be feasible, using even the term induced cancer stem cells (iCSCs)
iPSCs for modelling full-blown common cancers has been proposed in this regard (Czerwińska
is presently beyond our reach; in fact, we do not et al. 2018). But even this notion might be
understand the biology of such tumours in such a plagued with a not negligible naivety – we have
detail that would allow us to reproduce them in seen that although pluripotency transcription
form of gene-modified iPSCs derived from nor- factors do participate in cancer stemness, this is
mal cells of the respective patients. Although we just one piece in the complex mosaic of biological
have catalogued thousands of cancer-specific mechanisms underlying cancer stem cells. We are
mutations and we have tools on how to introduce far from denying the potential of pluripotency
them with great precision into the genome of reprogramming for cancer modelling, but in our
iPSCs, we are still dramatically remote from view, this would provide just one of many exper-
being able to reproduce cancer genome and imental models, and only their combined use
epigenome and complex biological behaviour. hopefully provides us with crucial new knowl-
Not only that a lot of oncogenic mutations, well edge in order to combat this devastating disease.
characterized in isolation, act with pronounced
context dependency, but perhaps the major hurdle Acknowledgement This work was supported by the
is the cellular complexity of advanced tumours, Czech Science Foundation project no. 17-17636S. We
are grateful to Dr. Pavel Dvorak for help with the
with complex clonal heterogeneity and functional JUN-Sox2 immunofluorescence and Prof. Dr. Siegried
hierarchy. Moreover, this complexity is not static Schwarz for critical reading of the manuscript.
but constantly changes due to both genome insta-
bility of cancer cells and incessant reciprocal Conflict of Interest None
interactions with tumour microenvironment. In
this view, the endeavour to generate, starting Ethical Approval The authors declare that this article
from a patient-derived iPSCs, a “cancer-on-a- does not contain any studies with human participants or
dish” and use it as a screening platform within animals.
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https://doi.org/10.1007/5584_2021_656
# Springer Nature Switzerland AG 2021
Published online: 24 August 2021
101
102 M. Čater and G. Majdič
isolated from different tissues. These cells are and glia cells in vitro (Reynolds and Weiss
multipotent, meaning that they are capable of 1992; Ma et al. 2009). Neural stem cells, mesen-
differentiating into more than one cell type, but chymal stem cells, muscle stem cells, epidermal
not all cell types (Fortier 2005). Mesenchymal stem cells, and some others meet the basic criteria
stem cells, a special type of adult stem cells, for stem cells (Prockop 1997; Gage 2000; Watt
have become increasingly important in the field 2001) as they can proliferate and differentiate into
of regenerative medicine in recent years for various tissues in vitro, whereas corneal stem
treating certain human diseases (Giordano et al. cells and endothelial stem cells are only capable
2007; Trounson et al. 2011; Jossen et al. 2014). of differentiating into a single type of
They are being investigated for their therapeutic differentiated cell (Daniels et al. 2001; Verfaillie
potential in inflammatory, autoimmune, and 2002). The most studied adult stem cells are the
degenerative conditions in preclinical and clinical hematopoietic stem cells, while other adult stem
studies (Inamadar and Inamadar 2013; Ratcliffe cells were defined much later and are therefore
et al. 2013). less studied (Verfaillie 2002). In this chapter, we
Species and strain variations in the properties focus on adipose-derived, muscle, neural, and
of adult stem cells from different tissues and their spermatogonial stem cells.
requirements for optimal growth have been
reported in numerous publications although in
general, stem cells are difficult to isolate and 2.1 Adipose Tissue-Derived Stem
maintain in vitro (Baddoo et al. 2003; Peister Cells
et al. 2004; Sung et al. 2008). In addition to
growth medium composition and incubation tem- Adipose tissue is a rich source of mesenchymal
perature, partial oxygen pressure, extracellular adipose tissue-derived stem cells (Zuk et al.
matrix proteins, and contacts with other cells are 2010), which can differentiate into mesodermal
known factors that affect stem cell viability, pro- cells such as osteoblasts, chondrocytes,
liferation, function, and differentiation (Yoshida adipocytes, and muscle cells (Zuk et al. 2002;
et al. 2009). In this review, we describe the impor- Lee et al. 2004; Guilak et al. 2004; Lin et al.
tance of serum in the culture medium and the role 2008). Therefore, these cells have an important
of atmospheric oxygen in the growth and differ- potential for cell therapy and are used for
entiation of adult stem cells from various sources. immunomodulation in pathologies such as
A broad literature review is accompanied by our Crohn’s disease, regenerative medicine, and aes-
own studies. thetic medicine (Le Blanc and Ringden 2007;
Ringden et al. 2007; Abdi et al. 2008; Mirotsou
et al. 2011). Adipose tissue-derived stem cells
2 Sources of Adult Stem Cells offer several advantages over other cell types.
Adipose tissue is easily accessible, requiring min-
Mesenchymal stem cells, also called multipotent imally invasive surgery for its harvesting. In addi-
mesenchymal stromal cells, are found in adipose tion, adipose tissue contains many more
tissue, bone marrow, umbilical cord, and dental progenitor cells in comparison to other tissues
pulp (Klingemann et al. 2008). Muscles contain (e.g., bone marrow) (Strem et al. 2005).
skeletal muscle stem cells and hematopoietic stem Adipose-derived stem cells also have great
cells (Kawada 2001; Chen and Goldhamer 2003). expansion potential (Lee et al. 2004; Kern et al.
Spermatogonial stem cells can be isolated from 2006) which is important for cell therapies.
testes (Goossens and Tournaye 2006; Guan et al.
2006). Adult stem cells in the mammalian brain
(neural stem cells) were discovered much later 2.2 Muscle Stem Cells
than in other tissues (Altman and Das 1965;
Altman 1969), and it has been shown that these Muscle stem cells belong to the satellite cell pop-
neural stem cells can differentiate into neurons ulation and are responsible for skeletal muscle
In Vitro Culturing of Adult Stem Cells: The Importance of Serum and Atmospheric Oxygen 103
growth and repair. They reside between muscle throughout the brain but are found in specific
fibers within the basal lamina but outside the locations, especially in the ventricular-
muscle fiber (Montano 2014). Studies of muscle subventricular zone along the walls of the lateral
stem cells play an important role in the develop- ventricles and in the subgranular zone of the
ment of novel treatments for muscular disorders dentate gyrus of the hippocampus (Alvarez-
(Pomerantz and Blau 2008; Wang et al. 2014; Buylla and Lim 2004; Ma et al. 2005; Obernier
Relaix et al. 2021). However, these cells are diffi- and Alvarez-Buylla 2019). Lower number of neu-
cult to isolate and purify, so alternative myogenic ral stem cells is also found in the striatum, sep-
stem cells, including adipose-, bone marrow-, and tum, and spinal cord (Palmer et al. 1995; Weiss
umbilical cord-derived mesenchymal stem cells, et al. 1996b). Furthermore, the subcallosal zone,
as well as perivascular stem cells, are being located between the white matter and the hippo-
investigated for their potential as possible cell campus, has also been reported to contain adult
sources for treating muscle disorders (Pantelic neural stem cells (Kim et al. 2016). The genera-
and Larkin 2018). tion of neurons for cell therapy is promising for
the treatment of neurodegenerative diseases and
brain injuries.
2.3 Testicular Stem Cells
Spermatogonial stem cells reside inside the semi- 3 The Effect of Serum on Stem
niferous tubules within the testes. They are capa- Cell Growth
ble of self-renewal and producing daughter cells
to give rise to terminally differentiated cells, the For the successful proliferation of adult stem cells
spermatozoa. These cells are therefore responsi- in vitro and the maintenance of their stemness, the
ble for the lifelong maintenance of spermatogen- balance of nutrients and microenvironmental
esis (Nagano 2003; Kubota and Brinster 2018). conditions is of paramount importance. The cul-
Since the number of spermatogonial stem cells ture medium is one of the most important single
decreases with aging, aging of their niche is a factors in cell culture as it provides all essential
critical factor for the maintenance of these cells. nutrients (Butler and Jenkins 1989; Brunner et al.
Dysfunction of the niche leads to a decreased 2010). Serum is the most commonly used supple-
number of spermatogonial stem cells in older ment in cell culture. Fetal bovine serum (FBS) is a
men (Zhang et al. 2006). Spermatogenesis can common choice because it contains high
be impaired due to various congenital disorders, concentrations of growth factors and other impor-
resulting in male infertility (Matzuk and Lamb tant signaling molecules such as adhesion
2008). Spermatogonial transplantation can be proteins, nutrients, carrier proteins, cytokines,
used to restore fertility in infertile men and to and hormones that, along with its buffering
elucidate the mechanism of genetic defects in capacities, are required for cell survival, prolifer-
spermatogenesis (Shinohara et al. 2000). ation, and/or differentiation. FBS can be added to
the medium in varying amounts from 1% to 20%
to promote cell attachment and provide growth
2.4 Neural Stem Cells factors and vital nutrients (Harmouch et al. 2013;
Forcales 2015). However, the use of FBS in cell
The adult mammalian brain has a low regenera- culture can be problematic as FBS contains xeno-
tive capacity and is limited in its ability to replace geneic proteins and potentially pathogenic
neurons that become dysfunctional or atrophic microorganisms, which presents a risk for the
due to acute or chronic injury. The discovery of induction of an immunological response and the
neural stem cells opened the possibility of transmission of pathogens. FBS also poses a
harnessing them for endogenous brain repair problem for optimizing cell culture conditions
(Gage 2019). Neural stem cells are not distributed because its composition is not defined and
104 M. Čater and G. Majdič
80
60
40 MesenCult N
20 A20 N
0
0 1 2 3 4 5 6 7 8 9 10 11
time (days)
Fig. 1 Testicular stem cell growth in initial passage using serum-free MesenCult medium and A20 medium with 20%
FBS. Legend: N – normoxia
3.4 Neural Stem Cells actual oxygen content in tissues is much lower.
Interestingly, improved stem cell survival and
Standard methods for culturing neural stem cells reduced apoptosis have been reported when
were developed in the early 1990s and include the using low-oxygen partial pressure (Morrison
neurosphere method and adherent monolayer cul- et al. 2000; Studer et al. 2000). Several previous
ture (Reynolds and Weiss 1992; Palmer et al. studies have shown improvement of stem cell
1995; Ray et al. 1995). Protocols for isolation culture when cells were grown in oxygen
and in vitro cultivation of neural stem cells from concentrations below 10% (Guyton and Hall
adult mouse whole brain in the form of 1996; Carreau et al. 2011). This is believed to
neurospheres or monolayers use a serum-free better simulate in vivo conditions as oxygen
medium supplemented with growth factors like concentrations in the brain have been reported to
epidermal growth factor and fibroblast growth fac- be around 0.5% in the midbrain, 2–5% in the
tor, which are needed for the survival of cells cortex, and up to 8% in the pia mater (Mannello
(Walker and Kempermann 2014; Deshpande et al. 2011), about 3.8% in muscle (Carreau et al.
et al. 2019). However, in our study, very few 2011), and about 3% in the testes (Klotz et al.
neural stem cells were viable when cultured in a 1996). Interestingly, the oxygen concentration in
serum-free MesenCult medium, and they did not adipose tissue varies from 4.5% to 5% in lean
proliferate. Greater cultivation potential was mice to about 1–2% in obese mice (Ye et al.
achieved when FBS was available to the cells. 2007; Rausch et al. 2008; Netzer et al. 2015). It
Neural stem cells from all three brain regions has been suggested that local oxygen concentra-
(anterior, medial, and posterior) proliferated well tion may directly affect stem cell proliferation,
in A20 medium (Fig. 2). However, we observed a self-renewal, and differentiation. Stem cells
much slower cell proliferation in comparison to seem to benefit from residing in hypoxic niches
other stem cell types. It took 2 weeks for the neural where oxidative DNA damage can be reduced
stem cells to become about 80% confluent. They (Keith and Simon 2007). When stem cells are
were then transplanted into the next passage, cultured at oxygen concentrations that do not
where they continued to proliferate successfully. match those inside the niche microenvironment,
cells undergo a number of changes such as meta-
bolic turnover, oxidative stress, impaired motility,
4 The Effect of Hypoxia on Stem altered differentiation potential, and loss of
Cell Growth stemness potential (Mas-Bargues et al. 2019).
When cells are cultured at low oxygen concen-
Standard cell culture systems typically use envi- tration, any available oxygen diffuses into the
ronmental oxygen levels (20%) although the mitochondria, creating a hypoxic environment in
106 M. Čater and G. Majdič
80.0
60.0
N anterior
40.0
N medial
20.0 N posterior
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (days)
Fig. 2 Proliferation of neural stem cells from different parts using A20 medium with 20% FBS. Legend: N – normoxia
the cytosol. Hypoxia inhibits the activity of prolyl medium with FBS. Cells from all tissues (adipose
hydroxylases that regulate the activation of tissue, muscle, testes, and brain) grew much bet-
hypoxia-inducible factors. The original role of ter in A20 medium than in MesenCult medium
hypoxia-inducible factors, angiogenesis, has regardless of atmospheric conditions. However,
recently been expanded by new studies showing for cells grown in A20 medium, atmospheric
that they are also involved in self-renewal, conditions affected only proliferation of cells
stemness, and differentiation of stem cells. from adipose tissue and brain, while cells from
Under hypoxic conditions, hypoxia-induced testis and muscle tissue proliferated at similar
factors are not hydroxylated and are thus rates under both atmospheric conditions. Interest-
stabilized to initiate their transcriptional activity ingly, the effect of hypoxia was the opposite for
(Bell and Chandel 2007). Hypoxia-induced neural- and adipose tissue-derived cells. Cells
factors are also important for the regulation of from adipose tissue grew better under hypoxic
stem cell metabolism. Cells from hypoxic niches conditions, while neural cells grew better under
rely on anaerobic glycolysis to support ATP pro- normoxic conditions.
duction. When exposed to atmospheric oxygen
levels, cells are forced to decrease glycolysis
and increase oxygen consumption through mito- 4.1 Adipose Tissue-Derived Stem
chondrial oxidative phosphorylation. This meta- Cells
bolic switch affects cellular function as it
promotes senescence, genomic instability, and Many studies have observed low proliferation
shortening life span (Estrada et al. 2012). rate at the environmental oxygen concentration
In our laboratory we compared the growth of in adipose-derived stem cells (Efimenko et al.
adult stem cells from the BALB/c mouse in a 2011; Kim et al. 2012, Mas-Bargues et al.
serum-free and a serum-supplemented medium 2019). Our studies correspond to these findings
in normoxic (5% carbon dioxide, 20% oxygen, as the isolation of adult stem cells from the adi-
75% nitrogen) and hypoxic atmospheres (5% car- pose tissue of the BALB/c mouse using A20
bon dioxide, 2% oxygen, 93% nitrogen). Cells medium containing FBS was successful only in
from testis and muscle proliferated slightly better the hypoxic atmosphere, with cells visible as
under hypoxic conditions when grown in early as 24 h after tissue plating. Cell culture
MesenCult medium, but cells from adipose tissue reached 80–90% confluence by day 12 (Fig. 3).
grew only under normoxic conditions with this The cells were then transplanted and proliferated
medium, yet not as successfully as in A20 faster in the first passage than in the passage zero,
In Vitro Culturing of Adult Stem Cells: The Importance of Serum and Atmospheric Oxygen 107
80
60
40 MesenCult N
20 A20 H
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
time (days)
Fig. 3 Adipose tissue-derived stem cell growth curves in media in normoxic conditions and in MesenCult media in
zero passage using MesenCult and A20 mediums. Legend: hypoxic conditions
N – normoxia, H – hypoxia. Cells did not grow in A20
reaching 80% confluence in 4 days. The cells their proliferation slowed considerably after
were also transplanted in the second passage and reaching 50% confluence, and they never reached
reached 80% confluence in 3 days. Similar results 80% confluence. Spontaneous differentiation
for adipose tissue-derived stem cells have been occurred. On the other hand, oxygen concentra-
reported previously. Hypoxia has been shown to tion did not affect the proliferation of cells from
promote stemness, proliferative capacity, and via- muscles cultured in a serum-supplemented A20
bility of adipose tissue-derived stem cells and to medium as the cells grew at a similar rate under
prevent adipogenic differentiation through nega- both normoxic and hypoxic conditions. Prolifera-
tive gene regulation (Lin et al. 2006; Ye et al. tion was successful, and cell cultures reached
2007; Weijers et al. 2011; Yamamoto et al. 2013; about 80% confluence in 9–11 days (Fig. 4).
Choi et al. 2014; Kakudo et al. 2015). This likely Cells were transplanted in several more passages
reflects the adaptation of cells to low oxygen where they proliferated successfully under both
concentrations in adipose tissue in vivo. atmospheric conditions. This suggests that vari-
ous factors, including growth media, affect
the proliferation and self-renewal of stem cells
4.2 Muscle Stem Cells from different tissues, and cells from different
tissues show different sensitivity to oxygen
Unsuccessful proliferation of muscle stem cells concentrations. Although oxygen concentration
under normoxic conditions has been reported in affects proliferation of some cell types in vitro,
some studies (Csete et al. 2001; Lees et al. 2008). other factors, such as media and unknown tissue-
Ambient oxygen concentration affects cell cycle specific factors, seem to modulate the sensitivity
regulation as p53 phosphorylation increases in of cells to atmospheric conditions. Since we still
cultures grown at 20% oxygen, resulting in cell do not know all the factors that influence adult
cycle arrest (Chen et al. 2007). A similar effect of stem cells viability and stemness, future studies
atmospheric conditions was observed in our lab- need to focus on identifying factors that influence
oratory. Muscle stem cell isolation and prolifera- stem cells in culture in order to develop optimal
tion in a serum-free MesenCult medium was growth media and optimal atmospheric and other
successful only under hypoxia (Fig. 4), with conditions for culturing adult stem cells from
cells initially proliferating rapidly. However, different tissues.
108 M. Čater and G. Majdič
80
60
Mesencult H
40
A20 N
20 A20 H
0
0 1 2 3 4 5 6 7 8 9 10 11
time (days)
Fig. 4 Muscle stem cell growth in initial passage in both atmospheres using MesenCult and A20 mediums. Legend: N –
normoxia, H – hypoxia. Cell did not grow in MesenCult media in normoxic conditions
4.3 Testicular Stem Cells differentiation, while some report the opposite
(Vieira et al. 2011; Mas-Bargues et al. 2019).
Previous studies have shown that culturing sper- We found that neural stem cells grew faster in
matogonial stem cells can be improved by reduc- serum-supplemented A20 medium at 20% oxy-
ing the atmosphere oxygen concentration. Kubota gen in comparison to conditions with 2% oxygen.
et al. (2009) and Helsel et al. (2017) used 10% of With A20 medium, we obtained viable cells from
oxygen and achieved successful long-term cultur- all brain regions studied (anterior, medial, and
ing. Interestingly, however, no difference was posterior parts of the brain). However, overall,
observed in the proliferation rate under both cell proliferation of neural cells was slower
atmospheric conditions in our laboratory. We in comparison to other tissues. Cells grew in
compared the growth of adult murine spermato- both atmospheres, but proliferation was much
gonial stem cells in a serum-free and a serum- faster under normoxic conditions (Fig. 6). Under
supplemented media in normoxic and hypoxic normoxic conditions, cells were about 80%
atmospheres containing 20% and 2% oxygen, confluent in 2 weeks, whereas under hypoxic
respectively, and the results were similar with conditions, cells reached only about 40% conflu-
both media. Cells in serum-free media grew ence in the same time when isolated from the
very poorly under normoxic and hypoxic medial and posterior parts of the brain. This may
conditions, whereas cells in A20 medium grew reflect the higher requirement of neuronal cells
successfully regardless of atmospheric conditions for continuous oxygen supply. It is well known
(Fig. 5). that neurons require a constant supply of oxygen
and are the first cells in the body to die in hypoxia.
Therefore, it would be intuitive to expect neuro-
4.4 Neural Stem Cells nal cells to grow better in an atmosphere with
higher oxygen concentration.
The effect of oxygen concentration in the atmo- Indeed, this has already been shown as some
sphere on culturing neural stem cells remains studies reported that optimal growth of neural
unclear as there are many controversial studies stem cells occurs under normoxic conditions
published. Some studies of culturing neural stem (Kilty et al. 1999; Kang et al. 2010). In addition
cells report that ambient oxygen concentration to the medial part of the brain, which contains the
decreases their proliferation and promotes main regions known to harbor stem cells, we have
In Vitro Culturing of Adult Stem Cells: The Importance of Serum and Atmospheric Oxygen 109
80
60 MesenCult N
40 MesenCult H
20 A20 N
A20 H
0
0 1 2 3 4 5 6 7 8 9 10 11
time (days)
Fig. 5 Testicular stem cell growth in initial passage in both atmospheres using MesenCult and A20 mediums. Legend:
N – normoxia, H – hypoxia
70.0 N anterior
60.0
N medial
50.0
N posterior
40.0
30.0 H anterior
20.0 H medial
10.0 H posterior
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (days)
Fig. 6 Proliferation of brain stem cells from different parts in both atmospheres using A20 medium. Legend: N –
normoxia, H – hypoxia
successfully isolated stem cells from the anterior complex (Li et al. 2003; Bauer et al. 2005;
and posterior parts of the brain as well. Interest- Volkenstein et al. 2013; Völker et al. 2019),
ingly, all three brain regions appear to be an which likely explains our positive results in
equally good source of neural stem cells when obtaining stem cells from the posterior parts of
cultured with A20 medium in normoxia. The the brain.
stem cells from the anterior part of the brain Although there are some reports suggesting
most likely originated from the cortex and part that neural stem cells grow well also under hyp-
of the optic nerve as these areas have been previ- oxic conditions, this could be a response to path-
ously shown to contain stem cells (Palmer et al. ological conditions. Previous studies have shown
1999). Recently, it has been reported that neural that neural stem cell proliferation in the brain is
stem cells are also located in the inferior stimulated in vivo by hypoxia as a consequence
colliculus, auditory cortex, and dorsal vagal of stroke, asphyxiation, or other trauma as a
110 M. Čater and G. Majdič
homeostatic mechanism that attempts some adult stem cells require FBS in the medium,
neuroregeneration (Mannello et al. 2011; together with additional inducing factors, while in
Wagenaar et al. 2018). some cases, a serum-free medium is required. A
well-known example of heterogenous-induced
differentiation demands are adult stem cells
5 The Effect of Serum in Culture derived from adipose tissue, which can readily
Medium and Atmospheric differentiate into osteocytes, chondrocytes, and
Oxygen Concentration on Stem adipocytes, depending on the presence of certain
Cell Differentiation growth factors in the medium.
Fig. 7 Cells from adipose tissue, cultured for 3 weeks (hypoxia, MesenCult), spontaneously differentiated into
chondrocyte-like cells (left) and osteocyte-like cells (right)
survival of the cells, resulting in increased sponta- cells that can undergo neural differentiation.
neous chondrogenesis and osteogenesis. Neurospheres form when adipose-derived stem
Adipose tissue-derived stem cells can be induced cells are cultured at high density using a neurogenic
to differentiate into several different specialized cell differentiation medium, which is usually a serum-
types by altering the formulation of the growth free medium with the addition of antioxidants, indo-
medium (Zuk et al. 2002; Guilak et al. 2004). methacin, insulin, and isobutylmethylxanthine
Apart from additional induction molecules, osteo- (Safford et al. 2002, 2004).
genic and neural differentiation of adipose-derived
stem cells require a medium without FBS, whereas
adipogenic and chondrogenic differentiation media 5.2 Muscle Stem Cells
are supplemented with FBS (20% and 1–10%,
respectively) (Bunnell et al. 2008). Adipocyte dif- A high concentration of FBS in the medium is
ferentiation in vitro is induced with serum- required for the maintenance of undifferentiated
supplemented medium and induction cocktails adult muscle stem cells in vitro, with some studies
containing insulin, methylisobutylxanthine, hydro- also recommending the use of hypoxic conditions
cortisone or dexamethasone, and indomethacin or (Lees et al. 2008). This was confirmed in our
thiazolidinedione (Halvorsen et al. 2001; Bunnell study as cell proliferation was arrested when
et al. 2008). In addition, stem cells from adipose cells were cultured in serum-free medium in an
tissue can be induced to differentiate into ambient oxygen atmosphere. Moreover, muscle
chondrocytes or the osteogenic lineage. stem cells cultured in serum-free MesenCult
Chondrogenesis can be promoted by adding medium under hypoxia showed morphological
transforming growth factor, ascorbate, and dexa- changes after 9 days of cultivation with lipid
methasone to a serum-supplemented medium and vacuoles appearing inside the cells. Staining
maintaining the cells in a three-dimensional, with Oil Red O was performed and confirmed
rounded morphology in a micromass pellet culture that the cells accumulated lipid deposits and
or within a hydrogel (Erickson et al. 2002; Awad presumably spontaneously differentiated into
et al. 2003). Differentiation into osteoblast-like adipose cells (Fig. 8). No such spontaneous dif-
cells is induced by the absence of FBS in the ferentiation was observed in cells grown in A20
medium and by the addition of ascorbate, medium containing 20% FBS under normoxic or
β-glycerophosphate, and dexamethasone (Heng hypoxic conditions.
et al. 2004; Bunnell et al. 2008). Adipose tissue- For myogenic induction, cocultivation of mus-
derived stem cells can also serve as a source of stem cle stem cells with primary myoblasts and
112 M. Čater and G. Majdič
Fig. 8 Muscle-derived
cells, cultured for 9 days
(hypoxia, MesenCult),
spontaneously
differentiated into adipose-
like cells with rich lipid
deposits (stained with Oil
Red O)
additional induction with dexamethasone and MesenCult medium accumulated cellular lipid
FGF is usually used (Eberli et al. 2009; Bitto inclusions, like differentiated cells from muscle
et al. 2013). Stem cells isolated from adult muscle tissue. Differentiation into adipocytes was con-
can also be a good source of autologous neural firmed by Oil Red O staining (Fig. 9, left). How-
cells, useful for cell replacement in neurodegen- ever, under hypoxic conditions with 2% oxygen
erative and demyelinating diseases. When grown and when grown in the same serum-free medium,
in a serum-free medium under nonadherent cells appeared to spontaneously differentiate into
conditions, muscle stem cells can be induced to chondrospheroids, which was confirmed by posi-
differentiate into neurospheres. Further cultiva- tive staining with Alcian Blue (Fig. 9, right). This
tion under adherent conditions provokes differen- was only observed in cells isolated from testicular
tiation into neurons and oligodendrocytes tissue, and we do not know at the moment what
(Romero-Ramos et al. 2002). causes these differences in spontaneous differen-
tiation. The testis is composed of different cell
types, and in vivo, these cells have different
5.3 Testicular Stem Cells access to both oxygen and nutrients from the
blood due to the composition of the testis and
Serum in the growth medium is of high impor- the testis-blood barrier. It is therefore possible
tance for promoting the proliferation of undiffer- that different cell types grow better under
entiated spermatogonial stem cells. In our normoxic and hypoxic conditions and that these
laboratory, cells cultured in the absence of FBS cells have different differentiation capacities.
spontaneously differentiated. Cells isolated from Alternatively, the same cells could respond dif-
testes and grown in serum-free MesenCult ferently to different atmospheric conditions, but
medium under both normoxic and hypoxic this will have to be investigated in future studies.
conditions changed their morphology after only Induced in vitro differentiation of spermato-
4 days of cell culture. Interestingly, atmospheric genic cells seems to be a possible method for
conditions seem to influence the direction of the the treatment of male infertility. Studies of
spontaneous differentiation. Cells grown under in vitro spermatogenesis have shown that FBS
normoxic conditions at 20% oxygen in plays an important role and is essential to allow
In Vitro Culturing of Adult Stem Cells: The Importance of Serum and Atmospheric Oxygen 113
Fig. 9 Using MesenCult, testicular cells spontaneously differentiated in adipose (4 days, hypoxia, left) or chondrocyte-
like cells (9 days, normoxia, right)
the progress of spermatogenesis, together with factor (GDNF), brain-derived neurotrophic factor
additional molecules like growth factors and (BDNF), and nerve growth factor (NGF).
hormones (Lee et al. 2006; Sato et al. 2011; Neurotrophic factors and the presence of serum
Zhao et al. 2018). in the media are needed to induce differentiation.
The regulatory factors in the serum can strongly
influence the expression of neurotrophic factors.
5.4 Neural Stem Cells 1% FBS in the media reduces the expression of
GDNF in differentiating neural stem cells, while
A standard serum-free culture system for neural 10% suppresses it completely (Niles et al. 2004).
stem cells, also known as the neurosphere assay, A negative impact of ambient oxygen concen-
allows selective growth of stem cells isolated from tration on cell growth was shown also in the
the adult brain. Undifferentiated neural stem cells cultivation of neural stem cells. Cultivation
survive and proliferate, while most other under normoxic conditions can lead to spontane-
differentiated cell types die (Reynolds and Weiss ous differentiation of neural stem cells toward the
1992.) The use of certain growth factors such as glial lineage (Chen et al. 2007). However, some
EGF and FGF as mitogens can induce a consistent, studies report that normoxic conditions decrease
renewable source of undifferentiated neural stem proliferation and promote differentiation of neural
cells, which could be expanded into defined stem cells, while some reports suggest that low
proportions of neurons, astrocytes, and oligoden- oxygen concentration increases the differentiation
drocytes (Gritti et al. 1995, 1996, 1999: Reynolds potential of such cells (Vieira et al. 2011;
and Weiss 1996; Weiss et al. 1996b, 1996a). Mas-Bargues et al. 2019). Controversial results
Removal of growth factors, present in stemness- have also been observed in some other types of
promoting medium, and addition of FBS into the adult stem cells and can be explained partially by
growth medium cause neurosphere-derived cells to the concentration of oxygen in the atmosphere
differentiate (Rietze and Reynolds 2006; Liu et al. and the duration of exposure used in the studies.
2018). In general, undifferentiated neural stem Some of the studies used short-term hypoxia (less
cells express glial cell line-derived neurotrophic than 72 h), while others maintained the cells in
114 M. Čater and G. Majdič
hypoxia permanently. Furthermore, the oxygen reference to persisting neurogenesis in the olfactory
concentration in various studies varied between bulb. J Comp Neurol 137:433–457
Altman J, Das GD (1965) Autoradiographic and histologi-
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pare directly. maintaining germinal niches in the adult brain. Neuron
41:683–686
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Many studies have shown that adult stem cells Awad HA, Halvorsen YD, Gimble JM, Guilak F (2003)
isolated from different tissues respond differently Effects of transforming growth factor beta1 and dexa-
to different environmental conditions such as the methasone on the growth and chondrogenic differenti-
ation of adipose-derived stromal cells. Tissue Eng
content of growth media, in particular the pres-
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ence or absence of FBS, and atmospheric compo- Baddoo M, Hill K, Wilkinson R, Gaupp D, Hughes C, Kopen
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have a positive effect on both cell growth and the mal stem cells isolated from murine bone marrow by
negative selection. J Cell Biochem 89:1235–1249
prevention of spontaneous differentiation of stem
Bauer S, Hay M, Amilhon B, Jean A, Moyse E (2005) In
cells. Interestingly, cells from different tissues vivo neurogenesis in the dorsal vagal complex of the
often spontaneously differentiate when grown in adult rat brainstem. Neuroscience 130(1):75–90
serum-free media but follow different differentia- Bell EL, Chandel NS (2007) Genetics of mitochondrial
electron transport chain in regulating oxygen sensing.
tion pathways. In some cases, such as cells from
Methods Enzymol 435:447–461
the testes, even atmospheric oxygen concentra- Berger MG, Veyrat-Masson R, Rapatel C, Descamps S,
tion can affect the differentiation pathway of Chassagne J, Boiret-Dupre N (2006) Cell culture medium
cells. Apart from serum, the oxygen concentra- composition and translational adult bone marrow-derived
stem cell research. Stem Cells 24:2888–2890
tion in the atmosphere of the culture has a major
Bitto FF, Klimpp D, Lange C, Boos AM, Arkudas A,
impact on the stemness maintenance and survival Bleiziffer O, Horch RE, Kneser U, Beier JP (2013)
of stem cells, so this is an essential factor to Myogenic differentiation of mesenchymal stem cells
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Biomed Res Int 2013:935046
engineering and regenerative medicine.
Boscolo Sesillo F, Wong M, Cortez A, Alperin M (2020)
Isolation of muscle stem cells from rat skeletal
Funding Masa Cater was supported by Slovenian Minis- muscles. Stem Cell Res 43:101684
try of Education, Science, and Sport [Grant C3330-17- Brunner D, Frank J, Appl H, Schoffl H, Pfaller W,
529039] and by World Federation of Scientists Scholar- Gstraunthaler G (2010) Serum-free cell culture: the
ship in 2017/2018. Gregor Majdic is supported by ARRS serum-free media interactive online database. ALTEX
grant P4-0053 and J4-9436. 27:53–62
Bunnell BA, Estes BT, Guilak F, Gimble JM (2008) Dif-
ferentiation of adipose stem cells. Methods Mol Biol
456:155–171
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https://doi.org/10.1007/5584_2021_654
# Springer Nature Switzerland AG 2021
Published online: 17 August 2021
Keywords
T. Akkoc (*)
Genetic Engineering and Biotechnology Institute, Tubitak Allergy · Asthma · Mouse models
Marmara Research Center, Kocaeli, Turkey
e-mail: tolga.akkoc@tubitak.gov.tr
L. O’Mahony
Department of Medicine and Microbiology, APC Abbreviations
Microbiome Ireland, University College Cork, Cork,
Ireland
OVA Ovalbumin
R. Ferstl Der p Dermatophagoides pteronyssinus
Christine Kühne-Center for Allergy Research and
Education (CK-CARE), Davos, Switzerland Der f Dermatophagoides farinae
HDM House dust mite
C. Akdis
Swiss Institute of Allergy and Asthma Research (SIAF), SPF Specific pathogen free
Davos, Switzerland ASM airway smooth muscle
T. Akkoc PBMCs peripheral blood mononuclear cells
Department of Pediatric Allergy-Immunology, School of
Medicine, Marmara University, Istanbul, Turkey
119
120 T. Akkoc et al.
mice, there are also limitations due to complexity IL-13 and mediate several functions. These
of this disease. In other words, mice do not cytokines induce class switching of antibody
develop asthma. One can replicate important isotypes to the ε heavy chain for IgE antibody
components of the disease, but no single model production by B cells, development and recruit-
accurately models all the features of asthma ment of eosinophils, production of mucus, and
(Wenzel and Holgate 2006; Kumar and Foster reduction the threshold for contraction of smooth
2002; Ray et al. 2015). This is very important to muscle cells (Mosmann and Sad 1996;
take into account when choosing the correct Romagnani 1994; Corry 1999). Later, this
model to address the specific experimental ques- allergen-specific IgE binds to high-affinity IgE
tion. For example, chronic exposure models are receptors (FcεRI receptors) on the surface of
required to examine many of the structural mast cells and basophils. This series of activation
changes associated with inflammatory disease steps leads to the sensitization of patients to a
within the airways. This review focuses on the specific allergen. In the final phase, reexposure
different mouse models of asthma that are cur- to the sensitized allergens leads to activation of
rently in use and discusses the limitations of each effector cells and tissue injury occurs. The
model. degranulation of basophils and mast cells by
IgE-mediated cross-linking of receptors is crucial
in the type-I hypersensitivity reaction, which may
1.1 Immune Response to Allergens lead to development of type-IV hypersensitivity
chronic allergic inflammation. All these events
If immune tolerance is overcome to certain
require allergen-specific T cell activation in aller-
allergens, such as aeorallergens, food allergens,
gic individuals, while for healthy individuals
and insect venom, type I and type IV hypersensi-
peripheral, T cell tolerance prevents formation
tivity reactions ensue. Several factors, including
of allergic immunopathology (Hirose et al. 2017).
genetic susceptibility, the nature of antigen that
initiates the disease (antigen dose, time of expo-
sure, route of exposure, and structural
characteristics) and exposure to infections and 1.2 Mouse Models of Asthma
bacteria (Burks et al. 2008), influence the type
of immune response. Mice are the most common species studied for
Specialized antigen-presenting cells, such as animal models of asthma (Sagar et al. 2015). In
dendritic cells, have a crucial role in the presenta- particular, acute or chronic allergic lung inflam-
tion of antigenic compounds to T lymphocytes mation models utilizing ovalbumin (OVA) or
and lead to differentiation of naive T cells into house dust mite represent severe, persistent
Th1, Th2, Th9, Th17, and Th22 effector T-cell asthma models that have been successfully
subsets (Goldberg and Rock 1992; Akdis and established in mice. Usually, mice are systemi-
Akdis 2009). In murine models, the initial event cally sensitized to allergen with alum as an adju-
responsible for the development of allergic dis- vant via intraperitoneal injection and allergen
ease is the generation of allergen-specific challenged via the airways. The number of
CD4+Th2 cells (Jutel and Akdis 2011). The cur- sensitizations and challenges is decisive for the
rent view is that IL-4 stimulation promotes naive development of acute or chronic forms of this
T cell differentiation into Th2 cells (Romagnani model. The nature of the lung inflammation is
1994; Mosmann and Sad 1996; Abdelaziz et al. directly influenced by the genetic background of
2020). The allergic immune response occurs in the mice, the allergen, type of the sensitization
two main phases: first sensitization and develop- and challenge protocol, and contamination of the
ment of memory, and later followed by the effec- allergen with substances (e.g. LPS), which stimu-
tor phase and tissue injury. In the sensitization late the innate immune response (Zosky and Sly
phase, CD4+Th2 cells secrete IL-4, IL-5, and 2007) (Table 1).
122 T. Akkoc et al.
1.2.1 Genetic Background of Mice (Ewart et al. 2000), while C3H/HeJ and DBA/2
Various inbred mouse strains are available in mice are resistant to the development of allergen-
laboratories (Song and Hwang 2017). However, induced AHR (McIntire et al. 2001).
it is not possible to develop an asthma model with In many studies, either BALB/c or C57BL/6
all of these mouse strains. Based on the level of mice were used. BALB/c mice are known as
allergen-specific IgE and IgG1 production and the IgE-high responders to many allergens
degree of airway inflammation following (e.g. OVA, Bet v 1) and goat anti-mouse
repeated allergen challenges, high- and IgD-stimulation, whereas C57BL/6 mice are
low-responder mouse strains have been characterized by low-IgE response (Herz et al.
identified. Each different mouse strain shows a 2004b; Bousquet et al. 2000; McMillan and
different pattern of response following immuni- Lloyd 2004; Van Hove et al. 2009; Birrell et al.
zation to allergens. There are substantial 2003). In contrast, C57BL/6 mice exhibit rela-
differences in the ability to induce allergic inflam- tively more Th1-dominant immune responses
mation and AHR within these mouse strains. A/J compared to BALB/c mice and are utilized more
and AKR/J mice display high levels of allergen- often as colitis models (Van Hove et al. 2009)
induced AHR and reactivity to methacholine (Melgar et al. 2005).
Mouse Models of Asthma: Characteristics, Limitations and Future Perspectives. . . 123
a higher number of exposures through different relevant antigens such as HDM extract or grass
routes to achieve suitable sensitization (Blyth pollen have been used to develop a chronic
et al. 1996). Both OVA and HDM can be used asthma model (Fuchs and Braun 2008; Maunsell
as antigen to induce pulmonary inflammation. et al. 1968).
Inhaled HDM is more successful in developing
an respiratory allergy model because of the intrin-
sic enzymatic activity associated with the 1.3 Similarities and Differences
contents of this allergen (Bush et al. 2011). Between Murine Models
Extracts or purified major allergens of potent and Human Asthma
human allergens, including cockroach, ragweed,
or fungi, have been increasingly used as allergens Mice do not exhibit asthma-like lung inflamma-
in mice and other species (Barrett et al. 2003; tion, which is point-by-point comparable to
Kurup et al. 1997; Chapoval et al. 2002; Matsuda human asthma. There are important limitations
et al. 2021). After a sensitization period (usually that should be considered. Mice develop an
14–21 days), allergen challenge via airways is allergen-specific Th2-type immune response
carried out for several days. The administration using a highly artificial peritoneal allergen sensi-
of allergen through airways can be applied by tization procedure in the presence of an adjuvant,
nebulization, intratracheal (i.t), or intranasal such as alum (Debeuf et al. 2016). They do not
(i.n.) instillation. With these sensitization and develop spontaneous immediate and late phase
challenge protocols, mice develop certain features airway obstruction as is seen in human asthma
of clinical asthma, which can be defined as (Kumar and Foster 2002).
increased levels of allergen-specific and total There are significant differences in the devel-
IgE, eosinophil-dominant inflammatory cell infil- opmental features of human and mouse lungs at
tration to the peri-bronchial area, goblet cell birth. While the human lung continues to develop
hyperplasia, basement membrane thickening, from months to years after birth, mouse lungs are
and smooth muscle cell hypertrophy and AHR already mature at birth (Lodrup Carlsen and
to specific allergens or methacholine (Takeda Carlsen 2001). In humans, the immature lung is
and Gelfand 2009). exposed to environmental antigens or agents,
Although an acute allergen challenge model and the immune response within the immature
displays many key aspects of human asthma, human lung is different compared to that of a
there are obviously limitations when one mouse lung (Greenough et al. 2004). From the
compares these models with asthmatic patients. histopathological view, there are also differences
To overcome these limitations, several research in the lungs of human and mice, and this further
groups have developed chronic allergen chal- affects the histopathological changes of chronic
lenge models in order to reproduce more of the asthma models (Persson et al. 1997; Kumar and
features associated with asthma, such as goblet Foster 2002). Although submucosal glands are
cell metaplasia, epithelial hypertrophy, abundant throughout the medium and large
subepithelial fibrosis, and smooth muscle hyper- airways of the human lung, they are only present
trophy, which are altogether referred to as airway in the trachea of mice. In the large airways of the
remodeling (Akkoc et al. 2001; Blyth et al. 1996). mouse, the bronchial epithelium is not as fully
In order to develop a chronic asthma model, low stratified as in humans. This results in differences
levels of allergen should be repeatedly in the immune response to inhaled antigens (Lilly
administered to the airways for periods up to et al. 2005). Human asthma is restricted to
12 weeks. In most of the experimental models, conducting airways, while in mice, parenchymal
OVA has been used as an allergen (Fernandez- and vascular components of the airways are more
Rodriguez et al. 2008; Temelkovski et al. 1998; affected (Kumar and Foster 2002)
Wegmann 2008; Akkoc and Genc 2020), and in The role of airway smooth muscle (ASM) and
some experimental models, environmentally myofibroblasts in the bronchi in bronchial
Mouse Models of Asthma: Characteristics, Limitations and Future Perspectives. . . 125
induction of sensitized Th2 cells, which further obstruction, and nonspecific airway hyperreactiv-
releases IL-4, IL-5, IL-9, and IL-13 (Hamid and ity (Zhu et al. 1999). In mice, IL-13 binds to its
Tulic 2009). These cytokines play a role in airway receptor, IL-13Rα2, either in soluble form in
eosinophilia, pulmonary lymphocytosis, and serum or membrane-bound receptor, which is
mastocytosis; goblet cell hyperplasia with epithe- expressed by smooth muscle, while human
lial cell proliferation; smooth muscle hyperplasia IL-13 binds only to the membrane isoform
and increased thickness; subepithelial fibrosis; (Chen et al. 2009). Thus, murine models have
isotype switching of IgE and release from B been very useful in the examination of the roles
cells; increased production of chemokines, played by specific cytokines in the initiation of
which attracts T cells, eosinophils, neutrophils, allergic-type pulmonary inflammation due to
and mast cells; and narrowing of the airways the availability of knock-out and transgenic
because of smooth muscle contraction (Hamid models. However, murine studies should be
and Tulic 2009; Holgate 2008; Akdis et al. 2011). complemented by findings in humans, which con-
Although allergy is correlated with Th2-type firm the relevance of the cytokine data.
responses, some studies revealed that there is also
a Th1 immune response in the lung of children
with asthma (Brown et al. 2003). It is not unusual 1.5 Humanized Animal Models
to see increased IFN-γ and IL-12 levels in human of Asthma
asthma (Kenyon et al. 2000). Similarly, diseases
which have been characterized as Th1-dominant A humanized mouse refers to a mouse which
disorders can also display features normally contains functional human genes, cells, tissues,
associated with Th2-type immune responses. For and/or organs. For this purpose, immunodeficient
instance, although Sarcoidosis is a Th1 disease, mice are used to receive human cells, because
increased levels of IL-13 are also seen, which they can easily accept heterologous cells due to
suggests a Th2 component to the disease (Hauber deficiencies in the host immune system. Severe
et al. 2003; Akdis et al. 2011; Meyer et al. 2010). combined immunodeficient (SCID) mice were
One possibility is that Th2 responses are required demonstrated to be a useful model to analyze
at the initiation phase of asthma, while the inflam- human pathogenic mechanisms (Alessandrini
matory response, which occurs during the exacer- et al. 2020). Due to an immune defect, these
bation phase, is not limited to Th2 responses but mice are unable to reject allogenic or xenogeneic
also requires Th1 and perhaps Th17 responses transplants (Mosier et al. 1988; McCune 1991)
within the lung. and show an absence of mature and functional T
IL-4 and IL-13 are prototypical Th2-type and B lymphocytes (Bosma et al. 1983). It has
cytokines, and several studies have revealed that been shown that SCID mice reconstituted with
they play a crucial role in the induction of murine human peripheral blood mononuclear cells
asthma. Induction of IL-4 and IL-13 via both the (PBMCs) from asthmatic patients developed a
intranasal and intratracheal routes enhances aller- specific human IgE response, pulmonary
gic airway disease (Venkayya et al. 2002). inflammatory-type infiltrate, human Th2 cytokine
Enhanced transgenic expression of IL-4 in the production, lung inflammation, and AHR when
airway epithelial Clara cells elicits an inflamma- exposed to specific allergens by inhalation (Duez
tory response characterized by epithelial cell et al. 1996; Pestel et al. 1994; Chiang et al. 1995;
hypertrophy, with the accumulation of Gagnon et al. 1995; Steinsvik et al. 1999). Thus,
macrophages, lymphocytes, eosinophils, and it is possible to reconstitute components of the
neutrophils without any airway hyperreactivity human immune system in mice by transferring
or goblet cell hyperplasia (Rankin et al. 1996; PBMCs resulting in a human–mouse chimera
Zhu et al. 1999). Selective pulmonary expression with a functional human antigen-reactive immune
of IL-13 promotes a eosinophilic inflammatory system (Herz et al. 1998; Duez et al. 2000). The
response, mucus cell metaplasia, airway newer generation of humanized mouse models
Mouse Models of Asthma: Characteristics, Limitations and Future Perspectives. . . 127
use SCID mice crossed to IL-2Receptor gamma therapeutic approaches. Murine models will con-
Knockout mice, so-called NOD or NSG mice tinue to provide important mechanistic clues,
(NOD.Cg-PrkdcSCIDIL2rgtm1WjI/SzJ), which while improved mouse models may be useful
are the more efficient immunocompromised strain for extending our understanding of the basic
(Shultz et al. 2007). These mice lack mature T, B, mechanisms underpinning asthma and for exam-
and NK cells. In addition, they are deficient in ining new therapeutic options.
multiple cytokine signaling pathways as well as
displaying many defects in the innate immune
system (Shultz et al. 1995, 2005). Since 2000,
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https://doi.org/10.1007/5584_2021_657
# Springer Nature Switzerland AG 2021
Published online: 31 July 2021
Abstract
Abbreviations
Regenerative medicine promises a bright
future where damaged body parts can be ACI autologous chondrocyte implantation
restored, rejuvenated, and replaced. The appli- CLC cholangiocyte-like cell
cation of regenerative medicine is interdisci- ESC embryonic stem cell
plinary and covers nearly all fields of medical GVHD graft-vs-host disease
sciences and molecular engineering. This HbF fetal hemoglobin
review provides a road map on how regenera- HbS hemoglobin S
tive medicine is applied on the levels of hESC human embryonic stem cell
cell, tissue, and organ and summarizes the hiPSC human-induced pluripotent stem cell
advantages and limitation of human pluripo- HLC hepatocyte-like cell
tent stem cells in disease modeling and regen- hPSC human pluripotent stem cell
erative application. HSC hematopoietic stem cell
HSCT hematopoietic stem cell transplant
Keywords HU hydroxyurea
Cartilage damage · Hepatobiliary disease iPSC induced pluripotent stem cell
OCT osteochondral transplantation
modeling · Kidney dysfunction · Organoids ·
PACI articulated articular cartilage
Pluripotent stem cells · Regenerative
medicine · Sickle cell disease implantation
SCD sickle cell disease
1 Introduction
135
136 Y. Wang and Y.-Y. Jang
science, applied mathematics, and various forms with other complications. It is estimated that
of engineering. From cell therapy to organ regen- more than 300,000 children are born with sickle
eration, regenerative medicine is bringing new cell anemia every year globally (Piel et al.
hopes to various untreatable diseases of the past. 2013b). The direct pathophysiological conse-
Aided by innovations in gene-editing techniques, quence of HbS polymerization is vaso-occlusion
a variety of regenerative interventions are being and hemolysis. Clinically, the disease manifests
developed to treat difficult diseases of various itself in various forms of acute and chronic
cellular and organ bases. Additionally, stem injuries. Some of the most common symptoms
cells have been successful in modeling a variety include swelling and painful episodes. These
of diseases. These disease models provide excel- symptoms are often associated with other
lent testing ground for drug design and develop- complications such as acute chest syndrome,
mental research. Human stem cell-derived tissues osteonecrosis, priapism, kidney injury, and stroke
are also being transplanted to animal models to (Williams and Thein 2018).
create chimeras for more integrated studies. We Despite the genetic simplicity of sickle cell
are witnessing the advent of the new era, the era anemia, treating this disease had been challeng-
of regenerative medicine. In this review, we high- ing. Currently, there two FDA-approved
light the application of regenerative medicine in treatments for SCD: hydroxyurea (HU) and
treating three different diseases of cellular, tissue, L-glutamine. Doctors are often reluctant to pre-
and organ basis: sickle cell disease, articular car- scribe HU due to its associated misinformation
tilage damage, and kidney dysfunction. Addition- and poor adherence (Demirci et al. 2019). On the
ally, we highlight the groundbreaking other hand, L-glutamine treatments are extremely
advancements in human pluripotent stem cell dis- expensive and usually not covered by insurance.
ease modeling and discuss its current challenge Though these treatments can improve the
and limitation. patient’s quality of life, neither of them
completely cures the disease. Nonetheless, with
the advancement in newborn screening and vac-
2 Cells: Regenerative Medicine cination technology, more than 90% of SCD
for Sickle Cell Disease patients are expected to make it to their adult-
hood. It is expected that by 2050, there will be
The etiology of sickle cell disease (SCD) is well over 400,000 severe SCD patients (Piel et al.
understood for more than decades. Though it is 2013a).
the most common inherited hemoglobinopathy
disease, an effective cure is still lacking. SCD is
a monogenic disorder caused by a substitution 2.1 Hematopoietic Stem Cell
mutation in the beta-globin gene (HBB) of chro- Transplant
mosome 11. The mutation (A > T) at the sixth
codon of HBB gives rise to the sickling hemoglo- In 1984, a breakthrough was made in treating
bin, HbS. Structurally, the hydrophilic glutamic sickle cell disease using the concept of regenera-
acid is substituted with valine that forms a hydro- tive medicine. It was the first time that
phobic association with alanine, phenylalanine, hematopoietic stem cell transplant (HSCT) was
and leucine of the adjacent hemoglobin. Compar- performed on an HbSS patient. The patient was
ing to normal hemoglobin, HbS polymerizes rap- an 8-year-old girl who suffered acute myeloid
idly and significantly reduced the flow rate and leukemia at the same time (Johnson et al. 1984).
life span of red blood cells (Vekilov 2007). The source of transplant came from her
The most common form of sickle cell disease, HLA-matching sister. At the time, the procedure
HbSS, constitutes the homozygous mutation of was initially aimed to treat leukemia. However, it
rs334. HbSS patients express no normal hemo- also improved her condition of sickle cell anemia.
globin and show severe signs of anemia along It was not until then stem cell transplant has
From Cells to Organs: The Present and Future of Regenerative Medicine 137
become a therapeutic option for SCD (Salinas property of HbF was also confirmed in asymp-
Cisneros and Thein 2020). Currently, as of early tomatic SCD patients with hereditary HbF persis-
2021, 35 clinical trials are listed on clinicaltrials. tence (Forget 1998). Thus, SCD can be rescued
gov investigating allogeneic stem cell transplant by procedurally elevating HbF levels. It can be
as a treatment for sickle cell anemia. Among these done in two ways: enhancing the expression of
35 trials, 12 are already completed. Meanwhile, HbF by stimulating HbF upregulators and
four additional trials aim to use autologous stem knocking out HbF suppressor. A clinical trial
cell transplant with gene correcting interventions was launched in 2018 using the later approach.
to treat sickle cell anemia. Though each of these Lentiviral vectors carrying shRNA-inhibiting
clinical trials features a different perspective and BCL11A, a HbF silencer, were infused into
strategy, all of them follow the same rule of severe SCD patients with the hope of boosting
regenerative medicine: repair and replace the their HbF level (Demirci et al. 2019). The third
disease-origin cells. To date, HSCT is the only approach is to correct the pathologic mutation that
therapy that cures sickle cell disease. gives rise to SCD. It is the most straightforward
Hematopoietic stem cell transplantation yet most challenging approach. However, high
provides long-term benefits for SCD patients. hopes are given to the advancing CRISPR/Cas
However, the applicability is yet hindered by technology that can induce a double-strand
several obstacles. HSCT is limited by the avail- break at the sickling mutation and initiate repair
ability of HLA-matching donors. Only 15% of according to the provided template. Ideally, the
patients have such matches; on top of that, only patient’s cells would end up with homologous
10% undergo HSCT due to the associated risks copies of the normal beta-globin gene.
(Chakrabarti and Bareford 2007; Walters et al. Researchers are working hard to improve the
2001). It is possible to receive transplants for an specificity and efficiency of this technique.
unrelated HLA-matching donor. However, it dra-
matically increases the risk of complications such
as rejection and graft-vs-host disease (GVHD). 2.3 The Challenge of Gene Therapy
for SCD
Fig. 1 The regenerative treatments of sickle cell disease types and retransfused into the patient to replace the
The hematopoietic stem cells derived from sickle cell sickle cell
patients can be differentiated into improved hemoglobin
challenges will also imply the future of germline to develop advanced tissue engineering
gene therapy, potentially curing the patient’s technologies. These developments have brought
descendants. There will be consequential social, transformational changes to the treatments of
legal, and ethical issues based on gene editing. articular cartilage damage.
Articular cartilage injury can occur due to nor-
mal wear and tear or trauma. It is particularly
3 Tissue: Repairing Articular common in athletes and senior citizens. Patients
Cartilage Injuries who undergo arthroscopy are also likely to
develop cartilage injuries (Kalson et al. 2010).
The concept of regenerative medicine has The healing capability of cartilage injury is lim-
achieved great success in treating various diseases ited due to the lack of nerves and blood vessels in
on a cellular basis. In recent years, much attention the cartilage. Left untreated, articular cartilage
in regenerative medicine has been drawn injury could lead to osteoarthritis and cause
From Cells to Organs: The Present and Future of Regenerative Medicine 139
various symptoms, including swelling, pain, and regeneration techniques (Fig. 2). Microfracture is
compromised joint movements. Nearly half of the a common bone marrow stimulation approach for
older citizens in the United States suffer from cartilage repair. First developed in the early 80s,
osteoarthritis in various degrees (Jiang et al. microfracture differentiates mesenchymal stem
2020). Economically, it has become a huge bur- cells from fibrous cartilage (Steadman et al.
den in medical expenses. The current treatments 2010). However, such technique is rather unsatis-
for articular cartilage injury are obstructed by the factory in repairing larger cartilage damages that
difficulty in binding the regenerated tissue to its are bigger than 2.5 cm2 (Jones and Peterson 2006).
surrounding environments (Muhammad et al. Additionally, elderly patients tend to heal slowly
2019). However, the new tissue-engineered carti- to these procedures. Another method often used to
lage brings unprecedented advantages to articular treat cartilage injury is osteochondral transplanta-
cartilage repair. tion (OCT) (Yamashita et al. 1985). OCT can use
either autologous or allogeneic sources of
osteochondral columns to fill in the defect sites
3.1 The Current Treatments of the cartilage. In the autologous case, the
and Limitations osteochondral columns are usually removed from
the non-weight-bearing sites and transplanted into
The current treatment strategy for articular the injured cartilage. This technique is often lim-
cartilage injury is mostly based on surgical ited by the amount of transplantable osteochondral
columns (Andrade et al. 2016). Thus, it is only free strategy. It is important to note that the cell-
suitable for small defects. On the other hand, allo- free strategy is, in fact, not free of cells. It indi-
geneic osteochondral tissue transplantation is more rectly uses stem cells, such as mesenchymal stem
available but risks disease transmission and is very cells, to regenerate cartilage without directly
expensive. Autologous chondrocyte implantation transplanting these mesenchymal stem cells into
(ACI) is an alternative technique that harvests the patients. Two subcategories fall under this
chondrocytes from the non-weight-bearing sites technique. The first induces cartilage regeneration
of the articular surface and transplants them into by stimulating bone marrow stem cells in situ
the damaged sites after in vitro expansion combined with a transplanted biocompatible scaf-
(Brittberg et al. 1994). However, this technique is fold. The second approach is to integrate mesen-
still hindered by several limitations, such as the chymal stem cell derivatives, including cytokines,
invasive surgical procedure and the limited num- various RNAs, etc., to the transplanted scaffold to
ber of available cells. Additionally, the in vitro stimulate the regeneration of cartilage. These “cell-
expansion of the harvested chondrocytes is prone free” strategies are still in the animal testing stage
to dedifferentiation. Articulated articular cartilage (Jiang et al. 2020). The last strategy commonly
implantation (PACI) is repairing the cartilage used to generate tissue-engineered cartilage is the
defect with the crushed allogeneic or autologous scaffold-free strategy. This strategy uses
cartilage particles (Lu et al. 2006). Comparing to chondrocytes spheroids for cartilage transplanta-
OCT, this technique requires less donor cartilage. tion. It is similar to ACI. However, the difference
However, like the previously described is that ACI uses cell suspensions. The advantage of
techniques, PACI is only suitable for small carti- the scaffold-free approach is that it avoids the
lage defects no larger than 3.5 cm2 (Jiang et al. problem of developing complex scaffolds that are
2020). often unavailable. It has been challenging to
develop ideal scaffolds that meet the various
criteria of transplantation. These criteria include
3.2 Tissue Engineering for Cartilage but not limited to promoting cell growth, biode-
Injuries gradable at an appropriate rate, and adhesive.
Chondrosphere® is an approved scaffold-free
Cartilage tissue engineering combines product that uses chondrocytes spheroid to repair
advancements in material science, biomechanics, cartilage damage. The phase III clinical trial has
biochemistry, and cell biology. It has shown great demonstrated that Chondrosphere® is at least as
promise in regenerating hyaline cartilage and effective as microfracture in patients with small
repair the entire cartilage defect. Engineered tissue cartilage effects (Armoiry et al. 2019). In patients
based on mesenchymal stem cells displays excel- with cartilage defects bigger than cm2,
lent proliferation potential, differentiation ability, Chondrosphere® is proven to be a more effective
and low immunogenicity (Harrell et al. 2019). treatment.
Three main strategies are currently in use to gen- To make tissue-engineered cartilages more
erate tissue-engineered cartilage (Fig. 2). The first available and practical, we must overcome sev-
strategy is by establishing a cell-scaffold construct. eral obstacles. These obstacles include producing
The scaffold is often made of biocompatible an ideal scaffold that promotes adhesion and
materials such as collagen matrix and hydrogel. growth of seed cells, optimizing the differentia-
Chondrocytes and various types of stem cells are tion and expansion of cartilage-related cells, and
planted into these scaffolds before transplanting minimizing the tumorigenicity and heterogenicity
into the patients. The scaffolds offer support for of stem cells used. Also, the possibility of
the seed cells to differentiate and expand. Once disease transmission and immune rejection of
completely integrated with the patient, these transplanted allogeneic cell sources must be
scaffolds slowly degrade. Another approach to addressed and prevented. Fundamentally, we
generating tissue-engineered cartilage is the cell- need to grab a more detailed understanding of
From Cells to Organs: The Present and Future of Regenerative Medicine 141
how cartilage developments are regulated in vivo. Nishinakamura 2017; Takasato et al. 2015).
Many interactions of immune responses between These protocols use a combination of small
secreted factors, synovial fluids, and exosomes molecules and growth factors to direct a stage-
are yet to be determined. These interactions are specific differentiation similar to the development
the key factors to consider when optimizing the of embryonic kidneys. Renal structures including
physical property of tissue scaffold and nephron, glomeruli, interstitium, and collecting
maintaining a local environment ideal for ducts are self-organized in these organoids. How-
recovery. ever, the functional capability of these cultures is
often no match to their natural counterparts.
Advanced understanding of how kidney develops
4 Organ: Kidney Regeneration in vivo are in need to optimize these differentia-
tion protocols. Additionally, three major
Whole organ regeneration remains challenging at obstacles block the reality of renal organoid
the current stage of regenerative medicine. How- replacement therapy. These obstacles are
ever, significant improvements toward kidney off-target cells, vascularization, and reproducibil-
generation have been made in recent years. The ity (Geuens et al. 2020). Kidney organoids
need for modern organ regeneration and trans- derived from established protocol may contain
plantation is on the global rise. Kidney is the up to 20% of the nonrenal cell population
most frequently transplanted organ in the United (Combes et al. 2019; Wu et al. 2018). These
States (Alachkar et al. 2011). Around 69,000 off-target cells increase in prevalence as the
kidneys are transplanted every year globally organoid gets larger and eventually disrupts the
according to WTO (Organization 2008). In integrity (Geuens et al. 2020). It remains unclear
2014, United States alone had nearly 16,000 kid- how these off-target cells come to place. The cells
ney transplants (Wragg et al. 2019). These large in this off-target population resemble many cell
numbers do not nearly meet the needs of organ types, including neuronal, muscle cells, cartilage,
shortage. The average waiting time for an avail- and anywhere in between (Bantounas et al. 2018;
able kidney in the United States is almost two and Morizane et al. 2015). The lack of vascular struc-
a half years (Wragg et al. 2019). This wait time is ture is another major difficulty in renal organoids.
much longer in other places of the world (Lee To date, there is no kidney organoid that shows
et al. 2019). More than 16% of patients die or patent vasculature. These organoids simply do
become too sick to receive the kidney transplant not have enough endothelial cells nor the proper
while they are waiting (Cassuto et al. 2010). The guiding cues from the vasculature. As organoids
extreme scarcity of kidneys has led to problems grow larger, cell death in the center mass becomes
beyond the field of medicine, such as trafficking, inevitable due to the lack of nutrients and waste
compensated donation, and the expansion of exchange. The third major problem is scaling.
black markets. Researchers around the world are Current kidney organoids are roughly 1/10,000
seeking ways to grow transplantable kidneys and of a single human kidney by nephron counts
devices that can mimic the function of kidneys. (Geuens et al. 2020). Automated systems for
generating these organoids are being constructed
and improved to scale up the liver organoids
4.1 Organoids production (Czerniecki et al. 2018). However,
without vasculature, these organoids cannot be
Kidney organoids derived from human pluripo- assembled into a functional liver.
tent stem cells (hPSCs) are given hopes to bring
the future of renal replacement therapy in regen-
erative medicine. Numerous protocols are 4.2 Wearable Artificial Kidney
established to differentiate hPSCs into kidney
organoids (Freedman et al. 2015; Morizane et al. As the regeneration of a whole kidney remains a
2015; Taguchi et al. 2014; Taguchi and tremendous challenge, dialysis is the mainstream
142 Y. Wang and Y.-Y. Jang
of current kidney replacement therapy. Though be generated from various adult cells and tissues.
dialysis provides the means to survive kidney The abundance of iPSC and its easy maintenance
failure, it requires significant changes in the offers an unparalleled advantage for developing
patient’s daily routine. Beyond its physical incon- disease models. At the same time, hPSCs are
venience, long sessions of dialysis are becoming a extremely flexible as they can theoretically
burden that affects the mental health of patients differentiate into any cell type of the human
(Pereira et al. 2017). In recent years, efforts are body. The differentiation protocol of hPSCs to
made into the development of wearable artificial many cell types are well established and publicly
kidneys. These wearable kidney devices are cur- available. The most common cell types
rently under clinical trials (Davenport et al. 2007; differentiated from iPSCs include dopaminergic
Gura et al. 2005; Lee and Roberts 2008). The neurons, motor neurons, astrocytes, oligoden-
portability of these devices allows the patient to drocytes, cardiomyocytes, hepatocytes, pancreatic
continue to work and travel. Typically, these β cell, and lung epithelial cells (Fig. 3) (Abo et al.
devices are a few kilograms in weight and 2020; Bianchi et al. 2018; Corbett and Duncan
operated by battery (Salani et al. 2018). Inside 2019; Ehrlich et al. 2017; Hallett et al. 2015;
the wearable artificial kidney, blood is Karakikes et al. 2015; Ma et al. 2018; Soubannier
anticoagulated and pumped through a polysulfide et al. 2020). Patient-derived iPSCs are often devel-
hollow-fiber dialyzer (Gura et al. 2009). The oped into the diseased cell types to identify the
dialysates are pushed in a rhythm that linkages between the patient genotype and disease
compensates for the peak and trough of the phenotype as the iPSC reprogramming preserves
blood flow as the dialyzer transmembrane the genomic integrity of the patient. In the past
oscillates. A blood flow of 100 mL/min is decade, iPSCs have been successful in modeling
achieved with this system. To avoid the risk of countless diseases of genetic defects. However, it
accidental disconnection, wearable artificial remains a challenge to use iPSCs to accurately
kidneys are advised to use catheters instead of model more complex diseases, such as cancer.
needles in a fistula. However, the consequences Nevertheless, human iPSC offers an unprece-
of continuous catheter use remain unclear (Salani dented alternative to disease modeling. Most
et al. 2018). Some technological improvements importantly, these cell and organoid-based disease
are still needed before the general application of models are compatible with high-throughput
wearable artificial kidneys. screening, providing a faster and more efficient
solution to drug discovery and pathology research.
Fig. 3 Human iPSC disease models of various organ and iPSCs preserve the genome of the patient and can be
tissue types further developed into 2D and 3D disease models of vari-
Patient-specific cells can be derived into iPSCs that are ous origins
capable of differentiating into all three germ layers. These
144 Y. Wang and Y.-Y. Jang
diseases are supported by primary cells, tissues, 5.2 The Current Limitation of iPSCs
as well as various animal models. However, in Regenerative Applications
major drawbacks of primary culture and animal
models include high cost and low availability. Three major challenges hinder the downstream
Alternatively, immortalized disease lines are applications of human iPSCs. These challenges
often too altered to accurately reflect their sup- include potential tumorigenicity, immunogenic-
posed physiology. Though useful in many ways, ity, and heterogenicity (Sharkis et al. 2012;
animal models of hepatology are neither close to Yamanaka 2020). The tumorigenicity of iPSC is
recapitulating hepatobiliary responses in humans. mainly caused by three different reasons: incor-
In recent years, much focus has been dedicated rect patterning, reprogramming factors, and
to creating hepatobiliary “disease in a dish.” In a genetic abnormalities. The fate of stem cells is
nutshell, this concept is enabled by the in vitro strongly influenced by their patterning along with
differentiation of patient-derived stem cells. Nor- other cell types. In the occurrence of incorrect or
mal stem cells can also be genetically altered to incomplete patterning, niche-specific stem cells
express enhanced disease phenotypes, depending within the transplant often end up forming
on the configurable culture conditions. The major tumors. One example is the emergence of neural
advantage of this approach is that these in vitro rosettes, which will maintain normal develop-
disease models can be abundantly available and ment when it is patterned toward the cortex.
high-throughput compatible. It holds the potential However, simple in vivo injection of the same
to dramatically reduce the time span of funda- cells leads to cancerous growth (Malchenko
mental research and drug discovery processes. et al. 2014). The tumorigenicity of iPSCs can
The differentiation protocols of human iPSC also derive from the intrinsic property of
to hepatobiliary cell types, including hepatocyte- reprogramming factors. The common factors
like cells (HLC) and cholangiocyte-like cells used to generate iPSCs, including ct3/4, Sox2,
(CLC), have been reported from various sources Klf4, c-Myc, all have reported roles in cancer
(Corbett and Duncan 2019; Liu et al. 2011; development. C-Myc is one of the most fre-
Sampaziotis et al. 2017; Tian et al. 2016b). quently discussed proto-oncogenes. iPSC-
These protocols follow a multistep and stage- associated chimeric mice often develop tumors
specific procedure involving a symphony of key due to the reactivation of c-Myc and other
cytokines and growth factors. When iPSCs are reprogramming factors (Yamanaka 2020).
derived from patients of genetic diseases, the Another challenge iPSCs are facing is immune
genetic linkages of disease progressions are rejection. It has been controversial whether autol-
retained in the differentiation of these iPSCs, ogous iPSCs are immunogenic. Zhao et al. has
allowing in vitro observation and drug testing. suggested that the immunogenicity of autologous
Additionally, by altering the chemical composi- iPSC is caused by its abnormal gene expression
tion of iPSC culture media, researchers are also (Zhao et al. 2011). Their research provides an
able to recapitulate the development of example of immune rejection of autologous iPSC
hepatobiliary conditions caused by environmental transplants. The teratomas formed in the iPSC-
factors, for example, drug-induced hepatotoxicity transplanted mice showed obvious signs of rejec-
and alcohol liver disease (Sirenko and Cromwell tion, such as T-cell infiltration. A more recent
2018; Tian et al. 2016a, c). Altogether genetic and study suggested that neoepitopes of autologous
environmental, iPSC models are an excellent tool iPSCs can also originate from de novo mutations
for studying hepatobiliary diseases. in the mitochondria (Deuse et al. 2019). However,
In the past decades, a list of hepatobiliary in most cases, autologous iPSCs grafts do not
diseases was successfully modeled by iPSC technol- trigger an immunogenic response. On the other
ogy, many of which have led to the discovery of hand, allografts of iPSCs are much more immuno-
promising therapy candidates. A list of these genic. Nonetheless, allogeneic approaches are
diseases and their phenotypes is included in Table 1. much preferred due to their low cost and high
From Cells to Organs: The Present and Future of Regenerative Medicine 145
production. Immunogenicity caused by allogeneic (Ying et al. 2008). The “ground state” of these
transplants is often mitigated by immunosup- lines characterize an undifferentiated morphology,
pressants. One limitation of immunosuppressants lower DNA methylation content, and greater
is that it is often a lifelong treatment, especially in potential to produce chimeric mice and germline-
organ transplants. However, it is possible to avoid competent ESCs. Heterogenicity is not unique to
excessive immunosuppressing medication when mice PSCs. Human ESC and iPSCs also have
the graft sites are immune-privileged, for example, troubles with heterogenicity. The example of
the central nervous system, the spinal cord, and heterogenicity in humans can be demonstrated by
the eye. the comparison of hESC and hiPSC lines. Such
Heterogenicity originates from the intrinsic comparisons revealed significant differences in
differences between the iPSC lines. These their gene expression, epigenetic status, and differ-
differences include their morphology, growth entiation potentials (Yamanaka 2012). On the
curve, gene expression, and propensity to differen- other hand, when 20 or more hESC and hiPSC
tiate into various cell lineages. The downstream lines are compared, it was shown that overlapping
applications such as modeling, drug testing, and variations do exist. In the attempts to overcome
therapies are hugely hurdled by heterogenicity. heterogeneity, researchers attempted to convert the
This problem was first addressed in mouse ESCs. “primed” state of hPSCs into a “naïve” state. Mul-
It was later revealed that heterogenic mouse ESCs tiple approaches have been taken to induce the
could be converted into a neutral “ground” state by naïve or ground state pluripotency in hPSCs
two defined kinases: MEK and GSK3 inhibitors (Yamanaka 2020).
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https://doi.org/10.1007/5584_2021_660
# Springer Nature Switzerland AG 2021
Published online: 7 October 2021
151
152 P. K. Sundaravadivelu et al.
based therapy in various diseased conditions forth (Okita and Yamanaka 2011; Brouwer et al.
and disorders. Even though ESCs can be 2016; Omole and Fakoya 2018; Saha et al. 2018b;
differentiated into different cell types belonging Haridhasapavalan et al. 2020).
to three germ layers (Lerou and Daley 2005), they One of the primary factors influencing
are not considered ideal for therapy because of reprogramming efficiency is the starting cell source
ethical issues and their inability to be used in (Okita and Yamanaka 2011). The type of cell used
autologous therapy. On the other hand, there are for reprogramming greatly influences the molecular
certain obstacles to using ASCs for cell therapy, and functional properties of the generated iPSCs
namely, age-dependent abundance, accessibility, (Polo et al. 2010). Fibroblasts are the most com-
difficulties in the isolation procedures, and lim- monly used somatic cell source for the derivation of
ited differentiation potential (Bang et al. 2016; iPSCs because of their availability and well-
Saha et al. 2018a). established cell culture protocols (Raab et al.
Circumventing all these limitations, a ground- 2014; Khazaei et al. 2017). But there are few inher-
breaking study was published in 2006, in which ent limitations associated with it to be used for
the researchers induced pluripotency in termi- efficient reprogramming. Firstly, fibroblasts are of
nally differentiated cells (fibroblasts) to achieve mesenchymal origin and iPSCs are of epithelial
a pluripotent-like state by introducing a combina- origin. Hence, the former has to undergo a
tion of transcription factors, Oct4, Sox2, Klf-4, mesenchymal-to-epithelial transition to be
and c-Myc (OSKM; popularly called Yamanaka reprogrammed successfully, during which the
factors), using a retrovirus gene delivery mesenchymal-associated genes are repressed and
approach (Takahashi and Yamanaka 2006). epithelial-associated genes are expressed (Raab
These cells are called induced pluripotent stem et al. 2014). Secondly, the reprogramming effi-
cells (iPSCs) and can be differentiated into cells ciency is dependent on the age of the donor from
of all three germ layers. Subsequently, the first whom the cells are isolated and the passage number
human iPSCs were reported that utilized of these isolated and expanded cells in culture
fibroblasts as a starting cell source using two (Streckfuss-Bömeke et al. 2013; Rohani et al.
reprogramming factor combinations [(i) OCT4, 2014). Thirdly, constant skin exposure to muta-
SOX2, KLF4, and c-MYC (OSKM; Yamanaka genic agents like UV rays creates some alterations
factors) and (ii) OCT4, SOX2, NANOG, and in the genome, affecting the quality and clinical
LIN28 (Thomson factors)] (Takahashi et al. applicability of the reprogrammed iPSCs (Gore
2007; Yu et al. 2007). Thereafter, studies reported et al. 2011; Young et al. 2012). Due to these
that any somatic cell isolated from a healthy sub- limitations, reprogramming efficiency and the qual-
ject or diseased patient could be reprogrammed ity of iPSCs generated is very low compared to
into iPSCs using integration-based and other cell types (Kim et al. 2008; Eminli et al.
integration-free approaches (Singh et al. 2015; 2009; Sun et al. 2009; Yan et al. 2010). Therefore,
Menon et al. 2016; Dey et al. 2017, 2021; different research groups explored alternate cell
Borgohain et al. 2019; Haridhasapavalan et al. types (hepatocytes, β-cells, melanocytes, muse
2019; Ray et al. 2021), opening the prospects cells, myoblasts, amniotic fluid cells, urine-derived
for biobanking and generating patient-specific epithelial cells, keratinocytes, dental pulp cells,
cells that can be used for personalized therapy, blood cells, FSCs, ASCs, and so forth) for
bypassing the concern of immune rejection (Okita reprogramming to overcome the limitations men-
and Yamanaka 2011). Although iPSCs have vast tioned above and generate quality iPSCs (Iida et al.
potential in personalized medicine, certain 2013; Yoo et al. 2013; Raab et al. 2014; Singh et al.
challenges have to be addressed to efficiently 2015; Menon et al. 2016; Saha et al. 2018a; Chahine
generate quality iPSCs, like choosing an ideal 2021; Disler et al. 2021; Rogers et al. 2021; Winder
starting cell source, reprogramming method, and Trokovic 2021; Jamal et al. 2021; Li et al. 2021;
reprogramming factors, culture conditions, Liu et al. 2021; Pellicano et al. 2021; Petzendorfer
overcoming reprogramming barriers, and so and Guillot 2021; Ray et al. 2021).
154 P. K. Sundaravadivelu et al.
Recent advances in iPSCs research have led to utilized as a starting cell source for cellular
the usage of tissue-restricted stem cells derived reprogramming, their advantages over other cell
from adult (called ASCs) and fetal (called FSCs) types, and critical barriers associated with their
tissues as promising cell sources for the genera- usage and applications.
tion of iPSCs. Both ASCs and FSCs have unique
characteristics, notably limited self-renewal, and
multi- or unipotent differentiation potential that 2 NSCs
render them easier to reprogram than terminally
differentiated cells. Secondly, these cells exhibit NSCs are one of the tissue-restricted stem cells
endogenous expression of one or more having multipotent characteristics considered for
Yamanaka/Thomson factors and lack the same the generation of iPSCs. Isolating and obtaining
for lineage-specific genes, thereby making them NSCs is a difficult and highly invasive proce-
more amenable to reprogramming. Third, the dure. NSCs are primarily localized in the
tissue-restricted stem cells have been proven to ependymal lining of the subventricular zone and
have epigenetic profiles similar to ESCs than subgranular zone of the hippocampal dentate
mature cells (Hochedlinger and Plath 2009). gyrus as a discrete population in the adult brain
Therefore, these cells may require minimal epige- (Ma et al. 2009; Zhao and Moore 2018). They
netic reprogramming than mature cells. Fourth, have been isolated from the whole brain of adult
these tissue-restricted stem cells become indepen- or fetal mice. Importantly, human iPSCs were
dent of the expression of transgenes sooner than also generated by reprogramming NSCs derived
terminally differentiated cells (Eminli et al. from the fetus (Hester et al. 2009; Kim et al.
2009). Lastly, these cells may have accumulated 2009a) and adult (Xie et al. 2013). The brain
only a few genomic aberrations compared to tissues are minced or trypsinized in NSC media
matured cells and are therefore genetically stable and cultured until neurospheres are formed.
(Villa et al. 2004; Bernardo et al. 2007; De These neurospheres are replated and grown
Filippis et al. 2007; Zhang et al. 2007; Meza- until adherent NSCs appear (Fig. 1) (Kim et al.
Zepeda et al. 2008; Giorgetti et al. 2009; Wang 2009c; Tat et al. 2010). Even though isolation of
et al. 2019). All these reasons indicate that these NSCs is a highly invasive procedure, they have
tissue-restricted stem cells can be an ideal source been an efficient cell source for reprogramming
for efficient iPSCs generation. Due to these to iPSCs. NSCs derived from adult mice were
advantages, numerous studies have observed reprogrammed into iPSCs by retroviral transduc-
faster reprogramming kinetics and increased tion of four Yamanaka factors (OSKM) with
reprogramming efficiencies using tissue- high efficiency of 3.6% (Kim et al. 2008).
restricted stem cells as starting cell types for These cells can generate iPSCs 50 times more
reprogramming (Kim et al. 2008; Sun et al. efficiently than fibroblasts (Kim et al. 2008).
2009; Ge et al. 2012; Wang et al. 2019). iPSCs were also generated from the NSCs
In this chapter, we exclusively and compre- derived from the hippocampus tissue of
hensively discuss the most commonly used epilepsy patients with approximately 0.9%
tissue-restricted stem cells, namely, neural stem reprogramming efficiency using Yamanaka
cells (NSCs), hematopoietic stem cells (HSCs), factors (Xie et al. 2013).
mesenchymal stem cells (MSCs), and limbal epi- Numerous studies have reported that NSCs
thelial stem cells (LESCs), used in the generation endogenously express pluripotency-associated
of iPSCs along with spermatogonial stem cells genes, like high levels of SOX2 and c-MYC
(SSCs) possessing the inherent pluripotent trait. (Kim et al. 2009a) or high expression of SOX2
We provide a detailed overview of tissue- and modest expression levels of KLF4 and
restricted stem cells (isolated from adult and c-MYC (Hester et al. 2009). Apart from this,
fetal tissues; this chapter covers some of the stated SOX2 is the core transcription factor regulating
stem cells common to both adult and fetal tissues) the functional properties of NSCs (Shimozaki
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . . 155
Fig. 1 Human brain tissue can be digested and cultured to These stem cells form neurospheres, which can be subse-
derive a heterogenous cell population. From this popula- quently reprogrammed to generate iPSCs using
tion, NSCs can be isolated using NSC-specific markers. integration-based and integration-free methods
2014). Notably, adult mouse NSCs express using just three factors (OKM) (Eminli et al.
higher endogenous levels of SOX2 and c-MYC 2008).
than ESCs (Episkopou 2005; Kim et al. 2008). The expression of OK in NSCs yielded iPSCs
Therefore, by taking advantage of this endoge- with a reprogramming efficiency of 0.11% (Kim
nous expression, efforts have been made to et al. 2008; Silva et al. 2008), whereas the same
reprogram NSCs using fewer Yamanaka factors. with four factors (Yamanaka factors) introduction
NSCs were successfully reprogrammed using was 3.6% (Kim et al. 2008). Later, iPSCs were
three-factor combinations (OKM, OSK, OSM) also generated using the same Yamanaka factors
(Kim et al. 2008), or two-factor combinations but with a lower reprogramming efficiency of
[OK alone (Kim et al. 2008; Silva et al. 2008; ~0.13% (Tat et al. 2010). The variation in
Hester et al. 2009), or OM alone (Kim et al. reprogramming efficiency does reflect the
2008)], or just using O alone (Kim et al. region-specific cell isolation and its influence on
2009b, a). Apart from NSCs, the neural progeni- reprogramming efficiency. While the former
tor cells, which are the intermittent stage between study (Kim et al. 2008) used cells from the
multipotent NSCs and mature differentiated mouse brain per se, in the latter case, the cells
cells, are also reported to be reprogrammed to were taken from the subventricular zone of the
iPSCs (Eminli et al. 2008). These neural progen- mouse brain (Tat et al. 2010). However, the
itor cells have inherent SOX2 expression and, reprogramming efficiency with single factor O
therefore, can be reprogrammed successfully alone is very low (0.014% in mice and 0.004%
156 P. K. Sundaravadivelu et al.
in humans) (Kim et al. 2009b, a). Notably, the infections and a potential immune reaction.
single factor reprogramming eliminates onco- Thus, the derivation of iPSCs under animal-
genic factors like KLF4 (Ghaleb and Yang derived feeder-free conditions is crucial to gener-
2017) and c-MYC (Okita et al. 2007). Hence, it ate clinical-grade iPSCs for various biomedical
can circumvent the problem of tumorigenicity applications. Alternatively, NSCs can also be
when these iPSCs are employed for cell therapy. induced chemically to generate iPSCs using a
Notably, NSCs exhibit faster reprogramming cocktail of small molecules (Ye et al. 2016).
kinetics. The time taken for reprogramming Although the generation of iPSCs using this
mouse NSCs to iPSCs varies from one to reprogramming technique is slow, there is no
two weeks using Yamanaka factors (Kim et al. risk of integrating the transgene(s) in the genome
2008) or around 5 days using Yamanaka factors (Ye et al. 2016). Hence, further optimizations are
in the presence of two inhibitors (2i) and leuke- required to be worked out in employing a combi-
mia inhibitory factor (Silva et al. 2008). In con- natorial approach in using small molecules with
trast, mouse embryonic fibroblasts (MEFs) take or without fewer factors sans oncogenes to obtain
~3 weeks to be reprogrammed (Silva et al. 2008), iPSCs with higher efficiency.
indicating MEFs are much more challenging to
reprogram than NSCs. A decrease in
reprogramming efficiency and slower 3 HSCs
reprogramming kinetics was observed when a
lesser number of factors were used to reprogram HSCs are another source of tissue-restricted stem
for both human and mouse NSCs (Table 1). cells having multipotent characteristics, which are
Despite these limitations, using fewer factors is also well studied for reprogramming. HSCs can
advantageous since the delivery of few factors in differentiate into lymphoid and myeloid
mammalian cells is easier than delivering all four progenitors, which eventually give rise to mature
factors in separate vectors. Furthermore, it will blood cells (Eminli et al. 2009). HSCs can be
obviate the need for oncogenic factors KLF4 and isolated from various sources, namely, cord
c-MYC in the reprogramming cocktail and may blood, bone marrow, or peripheral blood, based
also reduce the chance of insertional mutagenesis. on the expression of cell-specific surface markers
Collectively, the endogenous expression of (Fig. 2). Like NSCs, isolating HSCs from bone
pluripotency-associated genes in NSCs makes marrow is also an invasive procedure, but they are
them ideal for reprogramming with a reduced more efficiently reprogrammed to iPSCs than
number of reprogramming factors and better differentiated mature blood cells (Eminli et al.
reprogramming efficiencies along with faster 2009; González et al. 2011). HSCs derived from
kinetics. mice were successfully reprogrammed with high
Furthermore, a study reported that NSCs can efficiency using a genetically homogeneous “sec-
attain and maintain pluripotency under feeder- ondary system” expressing the Yamanaka factors
free conditions and can proliferate for at least (Eminli et al. 2009). These HSCs gave rise to
20 passages (Choi et al. 2011). In this study, the iPSCs up to 300 times more efficiently than
reprogramming efficiency induced on the feeder mature T and B lymphocytes (Eminli et al.
layer was three times higher than that without the 2009). This study showed that the differentiation
feeder layer (Choi et al. 2011). However, the stage of the starting cell has a strong influence on
presence of feeders may increase the risk of non- the kinetics and efficiency of reprogramming.
human pathogen transmission and immune rejec- Moreover, cell proliferation is presumed to be
tion due to contamination from nonhuman crucial for efficient reprogramming (Jaenisch
antigens (Martin et al. 2005). The presence of and Young 2008). However, no apparent
unidentified pathogens, such as prions and viruses differences in reprogramming efficiency were
in these nonautologous feeder cells, could pose an observed between low proliferating HSCs and
increased risk of transferring pathogenic highly proliferating hematopoietic progenitors in
Table 1 Various studies that have reported the generation of iPSCs from NSCs
Reprogramming Reprogramming Reprogramming In vitro In vivo
Somatic cell source(s) Reprogramming factors technique efficiency (%) kinetics characterization characterization Reference(s)
Adult mouse NSCs OSKM Retroviral 3.6 0.5 1–2 weeks Yes Yes Kim et al. (2008)
OK transduction 0.11 0.02 2–3 weeks
Mouse NSCs OSKM (+2i + LIF) Retroviral 0.11 ~5 days Yes Yes Silva et al. (2008)
OK (+2i + LIF) transduction ~0.0125 2–3 weeks
Adult mouse NSCs O Retroviral 0.014 4–5 weeks Yes Yes Kim et al. (2009b)
transduction
Human fetal NSCs OK Retroviral 0.006 7–8 weeks Yes Yes Kim et al. (2009a)
O transduction 0.004 10–11 weeks
Human fetal NSCs OK Retroviral 0.01 3 weeks Yes Yes Hester et al. (2009)
transduction
Adult mouse NSCs OSKM Retroviral 0.13 0.06 ND Yes Yes Tat et al. (2010)
transduction
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . .
Mouse NSCs OSKM Retroviral 0.02 ~7 days Yes Yes Choi et al. (2011)
transduction
Adult human NSCs OSKM Retroviral ~0.2 18 days Yes Yes Xie et al. (2013)
transduction
Mouse NSCs VPA, CHIR99021, Small molecules ND 7–8 weeks Yes Yes Ye et al. (2016)
616,452,
Tranylcypromine,
Forskolin, Ch 55, EPZ,
DZNep
O OCT4, S SOX2, K KLF4, M c-MYC, NSCs Neural stem cells, 2i Two inhibitors, LIF Leukemia Inhibitory Factor, VPA Valproic acid, DZNep 3-Deazaneplanocin A, ND Not
Determined
157
158 P. K. Sundaravadivelu et al.
Fig. 2 Blood from human peripheral blood, bone mar- HSC-specific markers. These stem cells can be subse-
row, or cord blood contains a heterogenous cell popula- quently reprogrammed to generate iPSCs using
tion. From this population, HSCs can be isolated using integration-based and integration-free methods
this study (Eminli et al. 2009). These results reprogramming factors are not expressed in
implied that reprogramming efficiency is inde- CD34+ HSCs (Kambal et al. 2011). Furthermore,
pendent of the proliferation rate of cells, and it long-term HSCs (LT-HSCs) are a scarce popula-
is the differentiation state, rather than the prolif- tion of cells within HSCs isolated from bone
eration rate, which influences iPSCs formation. marrow and peripheral blood. HSCs isolated
Human HSCs isolated from the cord blood or from cord blood will have one in 10 LT-HSCs
bone marrow were also used as a starting cell and are characterized by the expression of
source for iPSCs generation (Giorgetti et al. 15 unique markers, CD49f being the most promi-
2009; Chou et al. 2011; Kambal et al. 2011). nent one (Wang et al. 2019). CD49f has been
These cells were reprogrammed by using either reported earlier as a specific LT-HSC marker as
five (OSKM+LIN28) or three (OSK) or just two HSCs positive for this biomarker were highly
(OS) factors with comparable efficiencies capable of producing long-term multilineage
(Giorgetti et al. 2009). The CD133+ HSCs have grafts (Notta et al. 2011). The CD49f+ LT-HSCs
a high endogenous expression of KLF4 and are rare in peripheral blood compared to bone
c-MYC than keratinocytes and fibroblasts marrow (Wang et al. 2019). These CD49f+
(Giorgetti et al. 2009). In contrast, these LT-HSCs from human bone marrow and
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . . 159
peripheral blood are reprogrammed within two to disorders, which can be used as a model to study
three weeks of infection with significantly higher and understand the mechanism of those disorders
efficiency, close to 50%, which is the highest ever (Ye et al. 2009; Lin et al. 2015; Okumura et al.
reported using these cells (Wang et al. 2019). The 2019). In addition, immortalized lymphocytes
CD49f+ LT-HSCs were 10-fold and 100-fold (lymphoblastoids) are also used as an effective
more amenable to reprogramming than bulk somatic cell source for reprogramming (Chahine
peripheral blood CD34+ cells and skin fibroblasts, 2021). In summary, hematopoietic cells like
respectively (Wang et al. 2019). Besides high HSCs, hematopoietic progenitor cells, and
reprogramming efficiency, the incidence of lymphoblastoids are considered potential cell
somatic single nucleotide variations and indels sources for reprogramming. Although obtaining
were low in CD49f+ LT-HSCs compared to skin HSCs is difficult and LT-HSCs are a rarer popu-
fibroblasts (Wang et al. 2019). A summary of lation, these cells have been an excellent choice
various studies that have reported the generation for reprogramming to iPSCs with high efficiency
of iPSCs from HSCs is listed in Table 2. and fast kinetics.
Apart from the HSCs population (Lin /
CD34+/CD38 /c-Kit /low) isolated through con-
ventional mode, several research groups have 4 MSCs
used CD34+ cells isolated from cord blood and
bone marrow as a cell source for the generation of MSCs are multipotent stem cells of stromal origin
iPSCs using different reprogramming methods. and can be isolated from both fetal and adult
This cell population is heterogeneous as CD34 sources. They have been successfully isolated
is a standard cell surface marker for cells belong- from adipose tissue, hair follicles, dental pulp,
ing to hematopoietic lineage, including HSCs, bone marrow, amniotic fluid and tissues,
hematopoietic progenitor cells, and some mature Wharton’s jelly from the umbilical cord, and so
hematopoietic cells (Majeti et al. 2007). They forth (Fig. 3) (Phinney and Prockop 2007). These
serve as the most commonly used cell source for cells can differentiate into various cell types,
reprogramming besides fibroblasts because they namely, adipocytes, osteoblasts, chondroblasts,
are readily available and can be isolated using myocytes, hepatocytes, and neuroectodermal
simple procedures (Haase et al. 2009; Takenaka cells. MSCs are an attractive source of cells for
et al. 2010; Ban et al. 2011; Chou et al. 2011; Yu biomedical applications due to their
et al. 2011; Nishimura et al. 2011; Ohmine et al. multipotency, immuno-modulatory activity, and
2011; Meng et al. 2012; Okita et al. 2013; Su et al. ability to provide trophic support to HSCs
2013a; Ye et al. 2013). Although they are a het- (Phinney and Prockop 2007). These cells are
erogeneous population containing mature cells, also commonly used for reprogramming to derive
iPSCs generated using these cells did not possess iPSCs, apart from the tissue-restricted stem cells
V(D)J rearrangements, unlike that seen in the discussed above. The efficiency and kinetics of
case of mature lymphocytes for reprogramming reprogramming using MSCs or MSC-like cells
(Chou et al. 2011). Notably, iPSCs can be isolated from different sources are listed in
generated from these CD34+ cells using merely Tables 3 and 4.
two factors (OS) (Meng et al. 2012). Moreover,
reprogramming using these cells results in fewer
single nucleotide variations than fibroblasts 4.1 Adipose-Derived Stem Cells
(Su et al. 2013b). Furthermore, it has been (ADSCs)
shown that these CD34+ cells are functional and
can be successfully reprogrammed into iPSCs As the name suggests, ADSCs are stem cells
even after storage for up to 23 years (Ye et al. isolated from adipose tissue. An ADSC is defined
2009; Broxmeyer et al. 2011). Besides, they can as an MSC within adipose tissue with multipotent
also be used to generate iPSCs from patients with differentiation and self-renewal capacity. These
160
Table 2 Various studies that have reported the generation of iPSCs from HSCs
Somatic cell Reprogramming Reprogramming Reprogramming Reprogramming In vitro In vivo
sources(s) factors technique efficiency (%) kinetics characterization characterization Reference(s)
Mouse HSCs OSKM Lentiviral 15.8 ND Yes Yes Eminli et al. (2009)
transduction
CB HSCs OSKM Retroviral ND 12–15 days Yes Yes Giorgetti et al. (2009)
OSK transduction 0.45 0.27 12–15 days
OS ND 12–15 days
CB HSCs OSKM Lentiviral ND 9 days Yes Yes Kambal et al. (2011)
transduction
LT-HSCs from PB OSKM Sendai viral vector 44.5 4.1 2–3 weeks Yes Yes Wang et al. (2019)
and BM
O OCT4, S SOX2, K KLF4, M c-MYC, HSCs Hematopoietic stem cells, CB Cord blood, LT-HSCs Long-term hematopoietic stem cells, PB Peripheral blood, BM Bone marrow,
ND Not Determined
P. K. Sundaravadivelu et al.
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . . 161
Fig. 3 Human hair follicle, dental pulp, fat, bone marrow, markers. These stem cells can be subsequently
Wharton’s jelly, amniotic fluid, or amniotic tissue can be reprogrammed to generate iPSCs using integration-based
processed to derive a heterogenous cell population. From and integration-free methods
this population, MSCs were isolated using MSC-specific
cells are isolated from patients undergoing bariat- comparatively easier to derive, and proliferate
ric surgery through lipoaspiration and liposuc- more rapidly (Bunnell et al. 2008; Lee et al.
tion, which are considered relatively less 2014), these cells are also considered valuable
invasive procedures (Zuk et al. 2002; Guilak cell sources for the generation of iPSCs (Sun
et al. 2006; Sugii et al. 2011). The derivation of et al. 2009). Human ADSCs display high endog-
ADSCs is rapid and can be achieved in less than a enous expression of factors such as basic fibro-
week (Sugii et al. 2011). The yield of ADSCs blast growth factor, transforming growth factor
after the expansion is relatively high and is β1, Activin A, vitronectin, and fibronectin and
approximately 0.4 106 cells/ml of processed can serve as a feeder layer for pluripotent cells
lipoaspirate isolated from the adipose tissue (Sugii et al. 2010, 2011). Moreover, these cells
(Guilak et al. 2006). This vital alternative source express high alkaline phosphatase levels, KLF4,
of somatic cells can differentiate into adipogenic, and MSCs marker CD44 (Qu et al. 2012). They
osteogenic, chondrogenic, and myogenic lineages also express low levels of OCT4, NANOG, and
(Bunnell et al. 2008; Gao et al. 2021). Since REX1, enabling efficient reprogramming (Tat
ADSCs are multipotent, abundant, accessible, et al. 2010). Moreover, ADSCs possess an
162
Table 3 Various studies that have reported the generation of iPSCs from ADSCs
Somatic cell Reprogramming Reprogramming Reprogramming Reprogramming In vitro In vivo
source(s) factors technique efficiency (%) kinetics characterization characterization Reference(s)
Human ADSCs OSKM Lentiviral ~0.2 15–16 days Yes Yes Sun et al. (2009)
(with feeder) transduction
OSKM ~0.01–0.03 18–20 days
(feeder-free)
Mouse ADSCs OSKM Retroviral 0.25 0.11 7–10 days Yes Yes Sugii et al. (2010)
(with feeder) transduction
OSKM 0.4 2 0.17 7–10 days
(feeder-free)
Human ADSCs OSKM Retroviral 0.74 0.12 24 days Yes Yes Sugii et al. (2010)
(with feeder) transduction
OSKM 0.008 ND
(feeder-free)
Mouse ADSCs OSKM Retroviral 1.14 0.77 12 days Yes Yes Tat et al. (2010)
transduction
Porcine ADSCs OSKM Lentiviral 0.0376 0.000814 7 days Yes Yes Zhang et al. (2014)
(with feeder) transduction
OSKM 0.0153 0.00106 8 days
(feeder-free)
Buffalo ADSCs OSKM Retroviral ND 12 days Yes Yes Deng et al. (2019)
transduction
Human ADSCs OSKM Retroviral ND ND Yes Yes Mao et al. (2019)
transduction
Human ADSCs OSKM Sendai viral vector ND 21 days Yes Yes Zhou et al. (2020)
O OCT4, S SOX2, K KLF4, M c-MYC, ADSCs Adipose-derived stem cells, ND Not Determined
P. K. Sundaravadivelu et al.
Table 4 Various studies that have reported the generation of iPSCs from MSCs derived from different sources
Somatic cell source Reprogramming Reprogramming Reprogramming In vitro In vivo
(s) Reprogramming factors technique efficiency (%) kinetics characterization characterization Reference(s)
Human hair follicle OSKM Lentiviral ~0.001 25–30 days Yes Yes Wang et al.
MSCs transduction (2013a)
Human DPSCs OSKM Retroviral 0.1 2–3 weeks Yes Yes Yan et al.
(double infection) transduction (2010)
Stem cells from OSNL Lentiviral 0.07 2–3 weeks Yes Yes Yan et al.
apical papilla transduction (2010)
Human DPSCs OSNL Lentiviral 0.03 2–3 weeks Yes Yes Yan et al.
OSNL transduction 0.1 2–3 weeks (2010)
(double infection)
Stem cells from OSNL Lentiviral 0.02 2–3 weeks Yes Yes Yan et al.
exfoliated OSNL transduction 0.08 2–3 weeks (2010)
deciduous teeth (double infection)
Human DPSCs OSKM Retroviral ND 2–8 weeks Yes Yes Beltrão-
transduction Braga et al.
(2011)
Human DPSCs OSKM Sendai viral 1.3 13–18 days Yes No Pisal et al.
transduction (2018)
Human DPSCs OSKLmL + shp53 + inhibitors for Episomal vectors 0.19 13–18 days Yes Yes Chandrabose
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . .
(continued)
Table 4 (continued)
164
epigenetic and genetic landscape that is more feeder-independent conditions (Sun et al. 2009;
amenable for reprogramming than terminally Sugii et al. 2010, 2011) with comparable
differentiated fibroblast cells (Sun et al. 2009). efficiencies and reprogramming kinetics
Also, reprogramming ADSCs to iPSCs can (Table 3). Sun and colleagues reported that the
deliver higher efficiency than NSCs (Tat et al. generation of iPSCs from human ADSCs was
2010), urine cells (Lee et al. 2014), keratinocytes two times faster and 20 times more efficient than
(Sugii et al. 2010) and is far more efficient than fibroblasts with efficiencies of 0.01% on feeder-
fibroblasts (Sun et al. 2009; Sugii et al. 2010; Tat free Matrigel substrate to 0.2% on MEFs as feeders
et al. 2010). (Sun et al. 2009). Although reprogramming under
Besides humans, ADSCs have also been feeder-independent conditions takes more time
isolated from different species, namely, mouse, (18–20 days) compared to using MEFs as a feeder
buffalo, porcine, and so forth, and efficiently layer (15–16 days), it eliminates potential
reprogrammed into iPSCs using viral transduc- variability caused by using feeder cells and negates
tion of Yamanaka factors with reasonable the problem of contamination of cells from the
efficiencies (Sugii et al. 2010; Tat et al. 2010; feeder layer (Sun et al. 2009). Furthermore, using
Zhang et al. 2014; Deng et al. 2019). The average nonintegrative approaches like episomal plasmids
reprogramming time to give rise to human and under feeder-free conditions can yield iPSCs at
mouse iPSCs is 2.5 and 1.5 weeks, respectively higher efficiency than viral-based approaches
(Sugii et al. 2011). Notably, reprogramming (Lee et al. 2014). This eliminates any animal-
human and mouse ADSCs to iPSCs is 100-fold derived pathogen transmission risk and establishes
and 5-fold more efficient than human and mouse a Good Manufacturing Practices (GMP)-
fibroblasts, respectively (Sugii et al. 2010). Fur- compliant system to produce iPSCs for therapeutic
thermore, mouse ADSCs were most amenable to applications. A summary of various studies that
iPSC formation with an 8- and 38-fold improved have demonstrated the formation of iPSCs from
reprogramming efficiency than NSCs and MEFs, ADSCs is mentioned in Table 3. All these studies
respectively (Tat et al. 2010). A similar observa- suggest that ADSCs are ideal for reprogramming,
tion was made where porcine ADSCs were more especially under feeder-independent conditions.
efficiently reprogrammed than fibroblasts under
feeder-free and serum-free conditions (Zhang
et al. 2014). The inclusion of Leukemia inhibitory 4.2 Dental Pulp–Derived MSCs
factor, CHIR99021, and PD0325901 in the
medium resulted in the generation of naïve-like Dental pulp serves as another exciting source of
porcine iPSCs (Zhang et al. 2014). Moreover, MSC-like cells. Isolation of MSC-like cells from
another finding reported that buffalo ADSCs dental pulp is accessible and less invasive
were reprogrammed more efficiently into naïve (Beltrão-Braga et al. 2011). These MSC-like
iPSCs under hypoxic conditions (5% of oxygen), cells are capable of adipogenic and osteogenic
very likely through activated hypoxia-inducible differentiation (Huang et al. 2008). Additionally,
factor-1α, since they were adapted physiologi- these MSC-like cells are found to endogenously
cally to a microenvironment where the oxygen express some of the pluripotent stem cell markers
level was 1–5% (Deng et al. 2019). Moreover, like OCT4, NANOG, and REX1 (Huang et al.
iPSCs derived by reprogramming human ADSCs 2008). Therefore, these MSC-like cells are con-
have better osteogenic differentiation ability and sidered closer to pluripotent stem cells than other
are considered excellent for bone tissue engineer- MSCs and differentiated mature cells (Huang
ing (Mao et al. 2019). et al. 2008; Beltrão-Braga et al. 2011). Moreover,
Human ADSCs were efficiently reprogrammed MSC-like cells from dental pulp are immune
by viral transduction of Yamanaka factors under privileged, and therefore, iPSCs generated from
both feeder-dependent (Sun et al. 2009; Sugii et al. them will not be rejected by the host immune
2010; Mao et al. 2019; Zhou et al. 2020) and response (Gomes et al. 2010; Beltrão-Braga
166 P. K. Sundaravadivelu et al.
et al. 2011). Hence, these cells are considered an by using the STEMCCA system (Streckfuss-
ideal candidate for the generation of iPSCs. In Bömeke et al. 2013). Interestingly, iPSCs derived
fact, MSC-like cells isolated from dental pulp from these cells also displayed a significantly
have been successfully reprogrammed to iPSCs higher differentiation potential to spontaneously
using Yamanaka factors under feeder-free beating cardiomyocytes (Streckfuss-Bömeke
(Beltrão-Braga et al. 2011; Hamada et al. 2020) et al. 2013).
and xeno-free (Thekkeparambil Chandrabose
et al. 2018) conditions. Efficient reprogramming
was also achieved when MSC-like cells were 4.4 Hair Follicle–Derived MSCs
isolated from various dental sources, namely,
the dental pulp, apical papilla, and exfoliated Hair follicles are the most accessible tissue to
deciduous teeth, and were reprogrammed using obtain MSCs from the human body without any
either Yamanaka or Thomson factors. The invasive procedure. MSCs isolated from these
reprogramming efficiency increased approxi- human hair follicles are capable of osteogenic
mately 5-fold when the cells were transduced and adipogenic differentiation (Wang et al.
twice with an interval of 24 h compared to single 2013a). Moreover, they were negative for the
transduction (Yan et al. 2010). The iPSCs major histocompatibility complex, which means
generated from reprogramming of Dental pulp that iPSCs derived from these MSCs can bypass
stem cells (DPSCs) extracted from the natal immune rejection and may serve as an elegant
tooth using Sendai viral mode of transduction source for cell therapy applications (Wang et al.
have shown reprogramming efficiency higher 2013a).
than human fibroblasts (Pisal et al. 2018). The
same was also true in the case of retroviral and
lentiviral modes of transduction, as mentioned in 4.5 MSCs of Fetal Origin
Table 4. More interestingly, a subpopulation of
dental pulp cells exist which expresses 4.5.1 Amniotic Fluid–Derived MSCs
pluripotency-associated genes and surface Amniotic fluid is found inside the amniotic sac,
markers (SSEA4, OCT3/4, SOX2, NANOG, protecting and facilitating nutrient and gas
LIN28, CD13, CD29, CD90, CD105) when exchange between the fetus and the mother
cultured under specific culture conditions, termed (Fauza 2004; Roubelakis et al. 2012). This amni-
as dental pulp pluripotent-like stem cells (Atari otic fluid is collected by a minimally invasive
et al. 2012). These cells were also capable of procedure (using a syringe) called amniocentesis
embryoid body and teratoma formation, (Li et al. 2009; Roubelakis et al. 2012). Amnio-
validating their pluripotency (Atari et al. 2012). centesis is routinely performed as part of prenatal
The wide range of applications for iPSCs derived diagnosis (Fauza 2004; Roubelakis et al. 2012).
from DPSCs is reviewed in detail elsewhere Amniotic fluid cells (AFCs) or amniocytes repre-
(Jamal et al. 2021). sent a mixed population of cells of fetal origin
(Polgár et al. 1989; Turner and Fauza 2009). This
heterogeneous population of cells contains a sub-
4.3 Bone Marrow–Derived MSCs population of self-renewing, multipotent stem
cells, known as amniotic fluid–derived stem
Bone marrow is a major source of MSCs, and cells (AFSCs), contributing to ~1% of AFCs
these cells have been reported to show basal (Fauza 2004; De Coppi et al. 2007). These
expression of OCT4 and SOX2 genes. AFSCs express certain MSC-specific surface
Streckfuss-Bomeke et al. have demonstrated that markers, namely CD29, CD44, CD49e, CD58,
the bone marrow–derived MSCs exhibit higher CD73, CD90, CD105, and CD166 (Roubelakis
reprogramming efficiency (~0.12%) than et al. 2012; Qin et al. 2016; Slamecka et al. 2016)
fibroblasts (~0.06%) and keratinocytes (~0.03%) and hence can be easily isolated and purified
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . . 167
using the same (Fig. 3) (Pipino et al. 2014; Qin efficiencies and kinetics (Li et al. 2009, 2013;
et al. 2016; Slamecka et al. 2016; Sung et al. Wolfrum et al. 2010; Anchan et al. 2011; Fan
2020). Moreover, these AFSCs express et al. 2012; Drozd et al. 2015; Bertin et al. 2015;
pluripotency-related genes and markers such as Drews et al. 2015; Yan and Li 2017; Xing et al.
SSEA-4, CD117+ (c-kit), NANOG, OCT4, 2018; Park and Mostoslavsky 2018; Wang et al.
c-MYC, REX1, and SOX2 (Li et al. 2009; Qin 2020; Yi et al. 2021).
et al. 2016). Expression of all these genes and
markers makes them a more desirable target for 4.5.2 Amniotic Tissue–Derived MSCs
reprogramming. MSCs derived from amniotic tissues express
Certain advantages in using AFSCs as a pluripotency-associated genes, such as OCT4
starting cell source are that they have the least and NANOG (Miki et al. 2005; Ge et al. 2012).
genetic aberration induced by the environment Importantly, these cells do not undergo aging and
and also have a distinct epigenetic and genetic are functional even after propagation for >2 years
aspect that aids in the acquisition of complete in culture (Walther et al. 2009). In addition, these
reprogramming (Fan et al. 2012; Li et al. 2013; cells possess immunomodulatory properties,
Yan and Li 2017). More importantly, since these where they can block the immune response in
cells are not prone to any mutations because of the host. Therefore, iPSCs generated from these
external factors, patient-specific iPSCs can be MSCs also render immunomodulatory properties
generated from them and used for autologous and can be used for therapeutic applications
gene therapy (Li et al. 2009; Drozd et al. 2015; (Ge et al. 2012).
Xing et al. 2018; Wang et al. 2020). A detailed
perspective regarding the usage of AFSCs for 4.5.3 Wharton’s Jelly–Derived MSCs
reprogramming has been reported earlier (Guillot Wharton’s jelly is a gelatinous tissue found inside
2016; Petzendorfer and Guillot 2021). Table 4 the umbilical cord containing connective tissues
enlists the approaches used by various groups (Stefańska et al. 2020). These tissues are usually
for reprogramming AFSCs to iPSCs (Pipino discarded after childbirth; therefore, their usage
et al. 2014; Qin et al. 2016; Slamecka et al. does not pose any ethical concern (Miere et al.
2016; Sung et al. 2020). These cells were 2014). Wharton’s jelly contains MSCs capable of
reprogrammed with efficiencies comparable to adipogenic, chondrogenic, and osteogenic differ-
fibroblasts but with shorter reprogramming kinet- entiation and display immunomodulatory
ics (Pipino et al. 2014; Sung et al. 2020). Since properties (Stefańska et al. 2020). Hence, iPSCs-
they already express certain pluripotency- derived from these MSCs from Wharton’s jelly
associated markers, reprogramming was carried may also bypass the host’s immune system
out using lentiviral transduction with fewer (Miere et al. 2014). Moreover, MSCs from
factors like OS or O alone (Qin et al. 2016). Wharton’s jelly are easily attainable and in more
Interestingly, human AFSCs isolated during the significant numbers than MSCs from cord blood
first trimester of pregnancy could form embryoid (Fu et al. 2006; Cai et al. 2010). Because of easier
bodies within two to three weeks upon culturing availability and immunomodulatory properties,
in vitro, but not teratomas in vivo (Moschidou MSCs from Wharton’s jelly are considered as
et al. 2012). Therefore, to induce pluripotency in one of the potential sources for the generation of
these cells, histone deacetylase inhibitor (valproic iPSCs. Successful reprogramming of these MSCs
acid) was used in the culture media, which made to iPSCs has been reported using viral transduc-
them pluripotent, as confirmed by teratoma for- tion of Yamanaka factors (Cai et al. 2010; Miere
mation (Moschidou et al. 2012, 2013). Not only et al. 2014; Ababneh et al. 2020). Thus, MSC-like
AFSCs but heterogenous AFCs are also widely cells derived from various sources can be utilized
used as a starting cell source for the generation of to derive iPSCs efficiently. A summary of iPSCs
iPSCs. Numerous studies have used these AFCs derived from different types of MSCs is listed in
for iPSC generation with better reprogramming Table 4.
168 P. K. Sundaravadivelu et al.
Fig. 4 Limbal epithelial tissue can be isolated from the LESCs can be isolated using LESC-specific markers and
human corneoscleral rim of an eye and cultured to derive a reprogrammed to generate iPSCs using integration-based
heterogenous cell population. From this population, and integration-free methods
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . . 169
episomal plasmid approach and under feeder-free tubules (De Rooij 1998; de Rooij and
conditions (Sareen et al. 2014). Interestingly, Grootegored 1998). SSCs contribute to about
lower reprogramming efficiency using the 0.02–0.03% of the cells of testis (Tagelenbosch
lentiviral mode of transduction (~0.0005%) and de Rooij 1993). Primarily, the biological role
(Hayashi et al. 2012) has been observed com- of SSCs is the sustenance of male fertility by
pared to the episomal method of transfection being the only type of ASCs that can pass on
(~0.005%) (Sareen et al. 2014). Although the the genetic information to offspring (de Rooij
lentiviral method of transduction is considered and Grootegored 1998; Kanatsu-Shinohara and
more efficient than the episomal approach, the Shinohara 2013).
difference in reprogramming efficiency might be Notably, unlike somatic cells, SSCs possess
because the former study (Hayashi et al. 2012) inherent pluripotent characteristics similar to that
used limbal epithelial cells containing LESCs, of ESCs (Kanatsu-Shinohara et al. 2008; Huang
while the latter (Sareen et al. 2014) used limbal et al. 2009) and do not need the introduction of
epithelial cells which were enriched with LESC any exogenous factors for their reprogramming
population to derive iPSCs. However, the latter (Lee et al. 2018). As early as 1992, pluripotent
study did not specify the exact method of LESC cell lines have been established from primordial
enrichment. LESC-derived iPSCs reported better germ cells under specific growth conditions
persistence of epigenetic memory than iPSCs (Matsui et al. 1992). Almost a decade later, testes
generated from fibroblasts (Sareen et al. 2014). cells from neonatal mice and SSCs from p53
The phenomenon of retaining certain epigenomic knock-out neonatal mice were converted into plu-
characteristics from the starting cell source makes ripotent cells under a defined microenvironment
the differentiation ability of iPSCs biased towards (Kanatsu-Shinohara et al. 2004). Subsequently,
the starting cell source, known as epigenetic extensive research was carried out to generate
memory. Earlier studies have reported that this pluripotent cells from GSCs (Ko et al. 2009,
phenomenon is a common characteristic of iPSCs 2012; Kossack et al. 2009; Lee et al. 2018).
generated from other cell sources (Polo et al. Moreover, pluripotent cells expressing
2010; Hu et al. 2010; Ohi et al. 2011; Vaskova pluripotency markers like SSEA4, TRA-1-81,
et al. 2013). Comparatively, LESCs are easier to OCT4, SOX2, and early hESC markers like
obtain than other tissue-restricted stem cells. To TNAP have also been reported from a testis
date, only two studies have been reported to use biopsy sample. These cells formed embryoid bod-
LESCs as a starting cell source for the generation ies and maintained high telomerase activity
of iPSCs (Hayashi et al. 2012; Sareen et al. 2014). (Kossack et al. 2009). SSCs derived from both
More research is required to explore the applica- mouse and human testis have been successfully
bility of LESCs in reprogramming. reprogrammed into pluripotent cell lines as con-
firmed by in vitro and in vivo assays of
pluripotency (Ko et al. 2009, 2012; Kossack
6 SSCs et al. 2009). In fact, SSCs are one of the few
ASCs that display OCT4 expression (Kanatsu-
SSCs are germline stem cells (GSCs) in males, Shinohara et al. 2004; Ko et al. 2009), with
which can maintain their population by self- OCT4 required for their maintenance and coloni-
renewal and also differentiate into sperm during zation ability after transplantation (Kehler et al.
the adult male reproductive life by a process 2004; Dann et al. 2008).
called spermatogenesis (Tagelenbosch and de Numerous studies have shown the conversion
Rooij 1993; de Rooij and Grootegored 1998; Ko of these SSCs to pluripotent ESC-like cells.
et al. 2009; Kanatsu-Shinohara and Shinohara Firstly, the testis cells are cultured under GSCs
2013). They belong to a distinct cell subpopula- culture conditions, containing a cocktail of
tion within type A spermatogonia, which resides growth factors resulting in the formation of
on the basement membrane of the seminiferous GSCs colonies on top of the monolayer of
170 P. K. Sundaravadivelu et al.
testicular cells (Kossack et al. 2009). Then, these be due to decreased expression of pluripotency-
colonies are manually passaged and grown under associated genes like OCT4, SOX2, and
ESC culture conditions with a feeder layer until NANOG, and increased expression of differenti-
they give rise to ESC-like colonies. These ation genes with age (Azizi et al. 2016). More-
colonies are called germline pluripotent cells over, this might also account for varying
(gPSCs) (Fig. 5) (Kanatsu-Shinohara et al. 2004; efficiencies of SSCs derived from different aged
Ko et al. 2009, 2012; Lee et al. 2018). To generate mice to convert to gPSCs (Table 5).
high-quality, clinical-grade pluripotent cell lines, Isolation and culture of SSCs is a labor-
feeder-free approaches for expanding and produc- intensive process and requires expertise to iden-
ing gPSCs have also been developed (Choi et al. tify the appropriate colonies of GSCs (Ko et al.
2014; Lee et al. 2018). The efficiency of 2010). Unlike other cell sources, the clinical
converting SSCs into gPSCs varies depending application of SSCs can transcend issues of ethi-
on cell plating density with and without feeder cal concerns, immune rejection, risk of tumor
cells (Ko et al. 2009; Lee et al. 2018). Interest- formation, among others (Chen et al. 2020).
ingly, these mouse SSCs can attain pluripotency Most cancer patients have to undergo chemother-
only till a certain age of the mouse. A study apy, radiation, or both, which may result in infer-
demonstrated that SSCs derived from adolescent tility in patients. In such cases, cryopreservation
(about 7 weeks) mice or older are almost impos- of SSCs followed by their autologous transplan-
sible to reprogram (Azizi et al. 2016). This might tation into the individuals concerned will assist in
Fig. 5 Human testis tissue can be digested and cultured to These stem cells can be subsequently reprogrammed in
derive a heterogenous cell population. From this popula- the presence of growth factors and culture conditions to
tion, GSC colonies (containing SSCs) can be isolated. generate ESC-like gPSCs
Table 5 Various studies that have reported the generation of pluripotent cell lines from cells of testicular origin
Somatic cell source Reprogramming Reprogramming Reprogramming Reprogramming In vitro In vivo
(s) factors technique efficiency (%) kinetics characterization characterization Reference(s)
Neonatal mouse None NA ND 4–7 weeks Yes Yes Kanatsu-Shinohara
testis cells et al.(2004)
Adult p53-KO None NA ND ~4 weeks Yes Yes Kanatsu-Shinohara
mouse SSCs et al. (2004)
Adult mouse SSCs None NA 0.01 2–4 weeks Yes Yes Ko et al. (2009)
Human SSCs None NA ND ND Yes Yes Kossack et al. (2009)
Adult mouse SSCs None NA ND 2–4 weeks Yes Yes Ko et al. (2012)
Human testis None NA ND 2–4 weeks Yes Yes Lim et al. (2013)
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient. . .
(SSCs)
Bovine testis cells O Electroporation 0.3 15–21 days Yes Yes Wang et al. (2013b)
Neonatal mouse OSKM Induction of ND 10–12 days Yes Yes Bermejo-Álvarez et al.
SSCs transgenes (2015)
Mouse SSCs None NA ND 46–143 days Yes Yes Azizi et al. (2016)
Adult mouse SSCs None NA 0.000369 3–5 weeks Yes Yes Lee et al. (2018)
O OCT4, S SOX2, K KLF4, M c-MYC, SSCs Spermatogonial stem cells, KO Knock-out, NA Not applicable, ND Not determined
171
172 P. K. Sundaravadivelu et al.
Fig. 6 Various tissue-restricted stem cells can be isolated differentiated into desired cell type(s) and used for various
from patients and/or healthy subjects and can be biomedical applications
reprogrammed to generate iPSCs. These iPSCs can be
usually found in terminally differentiated cells, an ideal cell source under serum-free, feeder-free,
and (v) capable of efficient reprogramming due and xeno-free conditions using a non-genetic,
to endogenous expression of stem cell-specific non-viral method is highly required. More exten-
genes. To summarize, stem cells from various sive research to identify ideal cell sources that
fetal and adult sources have been proven to be will generate iPSCs retaining genomic stability
efficient somatic cell sources for the generation of with increased efficiency and kinetics is vital for
iPSCs. However, there are certain hurdles in biobanking and numerous clinical applications.
using them as a starting cell source, like availabil- This will help translate this promising technology
ity, highly invasive collection procedures, and to derive patient-specific iPSCs for biomedical
isolation; therefore, more research is required to applications.
make ASCs and FSCs an ideal cell source for the
derivation of iPSCs and employing the Acknowledgments We thank all the laboratory members
reprogrammed iPSCs for various therapeutic and of Stem Cell Engineering and Regenerative Medicine
(SCERM) for their excellent support. This work was
biomedical applications. supported by North Eastern Region – Biotechnology
Additionally, the critical requirement is to cre- Programme Management Cell (NERBPMC), Department
ate a GMP-compliant system for the generation of of Biotechnology, Government of India (BT/PR16655/
clinical-grade iPSCs. A rapid, reproducible, NER/95/132/2015) and IIT Guwahati Institutional
Top-Up on Start-Up Grant.
robust, and facile iPSC derivation protocol from
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182 D. Bironaitė et al.
there was no activation of neither apoptotic nor immune system activation sometimes can give a
necrotic pathways. However, levels of better further myocardial outcome or initiate worse
antiapoptotic mitochondrial Bcl-2 and heat pathological heart remodeling leading to the devel-
shock protein 60 (Hsp60) in PVB19-positive opment of DCM (McCarthy 3rd et al. 2000), while
biopsies were almost threefold lower than in subacute myocarditis is the most common cause of
PVB19-negative ones revealing impairment of DCM (Magnani and Dec 2006).
mitochondria. Altogether, data indicate that The human myocardium can be infected by a
persistence of PVB19 in myocardiums of wide variety of viruses such as adenovirus, entero-
nonischemic DCM patients can cause virus, parvovirus B19, hepatitis C virus, human
myocardial ECM remodeling through the herpesvirus type 6, Epstein–Barr virus, and others
MMPs, such as MMP1 and MMP2, and mito- (Bowles et al. 2003; Kühl et al. 2005b; Matsumori
chondrial impairment. The correlative analysis et al. 1999; Leveque et al. 2011; Mutlu et al. 2011).
of measured biomarkers suggested likely fur- However, in recent decades, a significant shift
ther activation of apoptotic cell death pathways from adenoviruses or enteroviruses to parvovirus
rather than fibrosis. Data also suggest that B19 (PVB19), as a most frequent cardiotropic
antiviral therapy could be beneficial for virus in endomyocardial biopsies (EMBs) of
PVB19-positive DCM patients by managing DCM patients, has been observed (Kühl et al.
further pathological myocardial remodeling. 2005b; Pankuweit et al. 2000). It was shown that
PVB19 may persist in 30 to 60% of healthy human
Keywords hearts and be of no importance in the pathogenesis
of idiopathic DCM (Lotze et al. 2010; Moimas
Apoptosis · Dilated cardiomyopathy ·
et al. 2012). The PVB19 in DCM myocardium
Fibrosis · Inflammation · MMP/TIMP · PVB19
was confirmed to be dominating, but its relevance
to future prognosis has been doubted (Kuethe et al.
2007; Zimmermann et al. 2010). However, our
1 Background previously published study confirmed the direct
involvement of PVB19 in the initiation of DCM
Viral infection is one of the leading causes of acute in mouse model (Bogomolovas et al. 2016). In
myocarditis with ensuing dilated cardiomyopathy addition, other studies also suggest PVB19 being
and heart failure that often requires heart transplan- a frequent cause of patients’ DCM damaging
tation (Mason 2003; Kawai 1999). The chronic myocardium (Leveque et al. 2011). Recently, the
presence of viruses in myocardium is less harmful association between PVB19 and DCM in children
but is also less investigated (Kühl et al. 2005a; has been shown: the presence of PVB19 in the
Tátrai et al. 2011). Currently, the most accepted cardiac allograft was associated with higher
models of acute viral myocarditis in human and adverse post-hypertensive events (Das et al.
animal models is divided into three phases: the 2020). The inconsistent findings may be
virus entry into the myocardium, particularly related to the different stages of investigated viral
endothelium cells (phase I), followed by an DCM – different intensities and/or durations of
immune system dysregulation, increased infiltra- intramyocardial viral infection might have differ-
tion of immune cells, and secretion of inflamma- ent outcomes.
tory cytokines (phase II), which leads to Parvovirus B19 is a single-stranded DNA
cardiomyocyte destruction, cardiac remodeling, virus and member of the Parvoviridae family,
and dilated cardiomyopathy (phase III) (Maekawa which is mainly residing and replicating in ery-
et al. 2007; Verdonschot et al. 2016). During the throid progenitor cells (Verdonschot et al. 2016;
first acute phase of viral myocarditis, the direct Gallinella 2013). PVB19 is known as the main
cardiomyocyte lysis can occur further facilitating cause of erythema infectiosum in children,
virus entrance, myocardium impairment, and car- arthropathy, transient aplastic crisis, or aplasia
diac dilation (Maekawa et al. 2007). The acute of red blood cells (Verdonschot et al. 2016;
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 183
Anderson et al. 1984). In myocardium, PVB19 fibrosis in human PVB19-positive and PVB19-
can enter endothelial cells (EC), cause negative DCM myocardial tissues and sera with
vasocontraction, and damage cardiac tissue or the purpose of evaluating which of these processes
activate an inflammation response of the immune could have the highest impact on the chronic
system (Yilmaz et al. 2008). The VP1 unique remodeling of DCM myocardium. The inflamma-
region (VP1u) of the PVB19 capsid has been tion level has been confirmed by the
reported to act as a major determinant of viral intramyocardial infiltration of T cells (CD3,
tropism for erythroid precursor cells (Mrzljak CD45Ro) and macrophages (CD68) and secretion
et al. 2010). Our recent publication demonstrated of TNF-α, IL-6, and CRB. The intrinsic apoptotic
that exposure of either human- or rat-derived ECs pathway and mitochondria impairment-related
to recombinant VP1u was not acutely cytotoxic markers (Bcl-2, Bax, caspase-9, Hsp60), extrinsic
but led to the upregulation of cellular stress apoptotic pathway-related markers (APO1/Fas/
signaling-related pathways (Rinkūnaitė et al. CD95, FasL, caspase-8), and executing caspase-3
2021). Data also suggest that high levels of as well as collagen I synthesis/degradation-
circulating PVB19 during acute infection can related markers (PICP, ICTP, TGF-β1, TIMP1)
cause endothelial damage, even without active were investigated both in PVB19-positive and
replication or direct internalization of virus into PVB19-negative DCM myocardium tissues and
the cardiac cells. Beside the endothelium, the sera. In addition, the levels of MMP1, MMP2,
PVB19-positive myocarditis can also increase MMP9, and MMP13, markers of ECM degradation
the levels of leukotrienes and prostaglandins and in EMBs, were evaluated immunocytochemically,
impair intramyocardial Ca2+ circulation and left while fibrosis and myocardial necrosis were
ventricle functioning (Duechting et al. 2008; investigated histochemically. Necrosis was also
Bock et al. 2005; Lupescu et al. 2006; Tschöpe evaluated by the secretion of high-sensitivity tropo-
et al. 2005). nin T (hsTnT). We hypothesize that persistent pres-
In addition, the inflammatory events in the ence of PVB19 in DCM patients’ myocardiums is
viral myocarditis were shown to be responsible not a benign phenomenon but negatively affects
for the changed heart energetical potential (Wei heart extracellular matrix and energetic supply-
et al. 2014), impaired balance between ECM related processes that can worsen functioning of
regulating matrix metalloproteinases (MMPs) PVB19-positive DCM myocardiums compared to
and their tissue inhibitors (TIMP) (Pauschinger the PVB19-negative ones.
and Schultheiss 2004), initiation of cell-mediated
autoimmune responses (Caforio et al. 2007), and
activation of myocardial cell apoptosis or necrosis 2 Methods
ultimately affecting the whole myocardium
(DeBiasi et al. 2010). Since the main ECM com- 2.1 Selection of the Patients
ponent in the cardiac tissue is collagen type I
(up to 85%) (Jugdutt 2003), impairment and/or The study group consisted of 32 consecutive
degradation of its synthesis can negatively affect patients (mean age 43.14 11.86 years) with
heart cell communication, initiate cardiomyocyte clinically suspected nonischemic DCM that
anoikis, and impinge contraction and proper heart were admitted to a tertiary referral center. All
tissue functioning (Frangogiannis 2020; Valiente- patients signed informed consent to include their
alandi et al. 2016; Johnston and Gillis 2017; data in the study, and all investigations
Michel 2003). However, what pathophysiological conformed to the principles outlined in the Decla-
processes are ongoing during persistence of ration of Helsinki. All patients were mainly of
PVB19 in DCM myocardium is still unclear and NYHA III groups (93–94%). All patients showed
little investigated. enlarged left ventricle (LV) associated with the
The main objective of this study was to investi- significantly impaired systolic function (left ven-
gate the inflammation, apoptosis, necrosis, and tricular ejection fraction (LVEF) was less than
184 D. Bironaitė et al.
analyzer (Roche Diagnostics, Indianapolis, Before ELISA measurements, all serum samples
Indiana). Brain natriuretic protein (BNP) was were thawed on ice, centrifuged at 12,000 g for
measured by two-step immunoassay in human 5 min, and, if necessary, appropriately diluted.
plasma using chemiluminescent microparticle
immunoassay (CMIA) technology referred as
Chemiflex according to the manufacturers’ 2.4 Separation of EMBs According
recommendations. Serum sample and anti-BNP- to the Expression of PVB19
coated paramagnetic particles were combined.
After incubation, samples were washed and com- The presence of various types of viruses (adeno-
bined with an anti-BNP acridinium-labeled conju- virus, herpes simplex viruses 1 and 2, varicella
gate. Samples were incubated and washed again zoster virus, Epstein-Barr virus, cytomegalic
and the chemoluminescence initiating mixture was virus, parvovirus B19, hepatitis C virus, entero
added. Resulting chemoluminescence reaction has virus, rubella virus and human herpes virus 6)
been measured by a chemoluminometer and was negligible and samples were eliminated.
expressed as relative light units (RLU). The dominating virus expression in human
DCM myocardiums determined by nested PCR
was PVB19 (86%); therefore, all patients’ EMB
2.3 Preparation of Endomyocardial samples were subdivided into two groups:
Biopsies (EMBs) and Blood PVB19-positive (n ¼ 14) and PVB19-negative
Samples (n ¼ 18).
Nested PCR primers were chosen for the
Right ventricular EMBs were obtained using a detection of viruses including adenovirus as in
flexible bioptome via the right femoral vein (Allard et al. 2001), herpes simplex viruses
(Cooper et al. 2007). Biopsies were taken from 1 and 2, varicella zoster virus, Epstein–Barr
the right interventricular septum of the patients virus, cytomegalic virus, parvovirus B19, hepati-
with confirmed absence of coronary artery dis- tis C virus, enterovirus, rubella virus as in
ease. Collected heart tissue biopsies were imme- (McIver et al. 2005), human herpesvirus
diately inserted into clean cryovials, carefully 6 (HHV-6A and HHV-6B, GenBank accession
labeled, and registered. The EMBs were stored nos. NC001664.2 and NC000898.1, respec-
at 70 C as retained biosamples. All biopsy tively), Kirsten rat sarcoma viral oncogene homo-
samples were carefully labeled and registered log (KRAS, GenBank accession no. NM033360),
before cryopreservation. EMB specimens were and ubiquitin C (UBC, GenBank accession
thawed on ice before analyzing, appropriately no. NM021009). Forward and reverse primers
prepared for the assays, and immediately for the nested PCR detection of HHV-6, UBC,
investigated. EMBs were subjected to conven- and KRAS were synthesized by Metabion Com-
tional histochemical and immunohistochemical pany (Martinsried, Germany). Used primers are
evaluation, ELISA assay, and DNA and RNA shown in Table 2. Forward primers for the second
extraction for the amplification of viral genomes. round of PCR were labeled with
In parallel to the EMBs, three serum-separating 6-carboxyfluorescein (FAM) at the 50 end.
(SST II) 8.5 ml tubes (BD Vacutainer®) were Genomic DNA and total RNA were extracted
collected for serum sampling from each patient. simultaneously using ZR-Duet™ DNA/RNA
Collected blood samples were kept at room tem- Miniprep kit (Zymo Research, Irvine, CA,
perature for 30–45 min (no longer than 60 min) to USA). RNA (1 μg) was reversely transcribed in
allow clotting. Samples were centrifuged for 20 μl reaction volumes using random hexamers
15 min at the manufacturer’s recommended and First Strand cDNA Synthesis Kit (Thermo
speed (1,000–2000 RCF). The upper layer was Fisher, Vilnius, Lithuania) according to the
carefully aspired, checked for turbidity, aliquoted vendor’s recommendations and diluted to 100 μl
into cryovials, labeled, and stored at 70 C. with deionized water after the reaction. All PCRs
186 D. Bironaitė et al.
Table 2 Primers for the detection of human herpesvirus 6 (HHV-6), Kirsten rat sarcoma viral oncogene homolog
(KRAS), and ubiquitin C (UBC)
Primer Sequence (50 – 30 )
HHV6-N1 forward ACCCGAGAGATGATTTTGCGTG
HHV6-N1 reverse GCAGAAGACAGCAGCGAGATAG
HHV6-N2 forward CATAGCAACCTTTTCTAGCTTTGAC
HHV6-N2 reverse TCTATAACATAAATGACCCCTGGGA
UBC-N1 forward TTCTTTCCAGAGAGCCGAAC
UBC-N1 reverse CCCATCTTCCAGCTGTTTTC
UBC-N2 forward TGGGTCGCAGTTCTTGTTTG
UBC-N2 reverse CCTTCCTTATCTTGGATCTTTGCC
KRAS-N1 forward CTTTGGAGCAGGAACAATGTCT
KRAS-N2 forward AATCCAGACTGTGTTTCTCCCT
KRAS-N1/N2 reverse TACACAAAGAAAGCCCTCCCC
were run on a Professional Standard thermocycler Germany) and anti-CD68 (DAKO Hamburg,
(Biometra, Göttingen, Germany) as described in Germany), anti-MMP1 (Spring Bioscience Corp.,
(Allard et al. 2001). KRAS and UBC detection USA), anti-MMP2 (Leica Biosystems Newcastle
was used to validate extraction of nucleic acids Ltd., UK), anti-MMP9 (Leica Biosystems
and was performed in parallel according to the Newcastle Ltd., UK), and anti-MMP13 (Novus
conditions for viral DNA and RNR, respectively. Biologicals Europe, Cambridge, UK).
Final PCR products were tenfold diluted and The intramyocardial inflammation was
analyzed by capillary electrophoresis on a diagnosed according to the criterion established
Genetic Analyzer 3130xl, using GeneScan™ by the European Society of Cardiology Working
600 LIZ™ Size Standard and Gene Mapper Soft- Group on Myocardial and Pericardial Diseases
ware v4.1 (Applied Biosystems, Foster City, CA, (an expert consensus group): immunohistochemi-
USA) for sizing PCR fragments. In the case of cal detection of significant focal or diffuse cellu-
positive result, the genomic DNA or RNR lar infiltration in the EMB (14 leucocytes/mm2
specimens of blood samples were also tested including up to 4 monocytes/mm2 with the pres-
and excluded from the EMBs. ence of CD3-positive T lymphocytes 7 cells/
mm2) (Caforio et al. 2007).
2.5 Histochemical
and Immunohistochemical 2.6 Digital Evaluation
Assays of EMBs of Histochemical
and Immunohistochemical EMB
The EMBs were fixed in 10% neutral buffered Staining
formalin and paraffin-embedded in a tissue pro-
cessor. To estimate the extent of fibrosis, the Inflammatory infiltrates in the biopsies were
EMB specimens were stained with connective immunohistochemically classified on tissue
tissue stain Masson’s trichrome according to the sections, according to the expression of CD3
standard protocol. The total cardiac fibrosis (T lymphocytes), CD45Ro (active-memory T
(including interstitial and perivascular) was lymphocytes), and CD68 (macrophages). The
assessed: keratin and muscle fibers were stained number of positively stained cells in each biopsy
red, whereas collagen was stained blue. sample was scored by a pathologist and expressed
Immunohistochemical staining was performed as number of positive cells/mm2.
using antibodies: anti-CD3 (DAKO Hamburg, Digital images from the experimental glass
Germany), anti-CD45Ro (DAKO Hamburg, slides were obtained using ScanScope Digital
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 187
Slide Scanner (Aperio, Vista, CA) at x20 magni- Sonopuls sonicator, kept 30 min on ice,
fication and archived on a devoted Spectrum centrifuged at 12,000 g for 15 min, aliquoted,
Server 11.1.0.751 (Aperio). Quality control of and stored at 70 C. Protein level of serum
the scanned images and all further analysis were and EMB samples was measured using a
performed using ImageScope V11.1.2.760 modified Lowry Protein Assay kit according to
(Aperio) and WebScope V11.1.0.756 (Aperio). the manufacturer’s recommendations (Thermo
A Genie algorithm was used to measure the Scientific Inc., USA). Absorbance was measured
extent of fibrosis, to calculate immunoassayed with a spectrophotometer (Asys UVM
cells, and to evaluate the area of immunohisto- 340 Microplate Reader UK – Biochrom Ltd.) set
chemically stained elements in the myocardium. at 750 nm. A bovine serum albumin (BSA) stan-
Genie (GENetic Imagery Exploration) is a pattern dard curve was made to determine the protein
recognition algorithm that distinguishes spatial concentration of each unknown sample. Protein
and morphological features based on structures concentration was expressed as mg/ml. The final
(classes) provided by the user. The algorithm concentration of estimated biomarkers by ELISA
was run for the whole slides, ignoring the number was expressed as ng/mg of protein.
of overlapped tissue sections on it – making the Levels of apoptotic pathway-related proteins
process fully automated. For more details on the in EMBs and serum samples were measured
Genie methodology to evaluate fibrosis, see our using specific biomarkers: to identify the intrin-
previous publication (Daunoravicius et al. 2014). sic/mitochondrial apoptotic pathway, the
For this study, specific Genie classifiers were antiapoptotic Bcl-2, the proapoptotic Bax, the
developed: (A) Basic tissue recognition Genie intrinsic apoptotic pathway initiating caspase-9
classifier v1 algorithm was used to identify were estimated (Elabscience Biotechnology Co.,
myocardial fibrosis. We have used only spatial Ltd., China). In addition, the mitochondrial func-
recognition, disabling the detection of morpho- tioning was measured by the release of
logical features. Total percentage of cardiac fibro- myocardial intramitochondrial heat shock protein
sis was adjusted to a total tissue area ignoring the 60, (HSP60) (Assaypro, Saint Charles, Missouri,
glass; (B) Genie pixel counting algorithm has USA). The executing apoptotic caspase-3 was
been used to measure MMP1, MMP2, MMP9, measured by Novus Biologicals Europe kits
MMP13, and PICP in myocardial biopsies. (Cambridge, UK). An extrinsic apoptotic path-
There were no suitable antibodies for the way biomarkers APO1/Fas/CD95 (Fas receptor),
histochemical ICTP investigation. The results FasL (Fas ligand), and extrinsic pathway
were shown as sum of weak/moderate/strong pos- initiating caspase-8 were assayed using Novus
itive and negative pixels. In parallel, the positive Biologicals Europe kits (Cambridge, UK).
pixels were counted empirically by the The marker of collagen synthesis procollagen I
pathologist. C-terminal propeptide (PICP) was measured
using Bio-Medical ELISA kit (Bio-Medical
Assay Co., Ltd., China), while the marker of
2.7 ELISA Assays in EMBs collagen degradation C-terminal telopeptide of
and Serums type I collagen (ICTP) was measured by the
Biotech kit (Shanghai BlueGene Biotech
Collected heart tissue EMBs were lysed in 100 μl Co., Ltd., China). The fibrosis stimulating
of RIPA lysis buffer (Thermo Scientific Inc., transforming growth factor beta 1 (TGF-β1) was
USA) supplemented with protease and phospha- estimated by the Invitrogen ELISA kit
tase mini inhibitor tablets, 1 mM PMSF, 1 mM (Invitrogen, Paisley, UK) according to the
Na2VO4, and 25 mM NaF according to the manufacturers’ recommendations.
manufacturer’s suggestion (Thermo Scientific The pro-inflammatory cytokines TNF-α, IL-6,
Inc., USA). EMB samples were sonicated at and IL-1β were assayed in serums by solid-phase,
10 mV for 2 5 s on ice using a Bandelin chemiluminescent immunometric assays using
188 D. Bironaitė et al.
also investigated by ELISA (Table 4). Data PVB19-positive versus PVB19-negative serums
revealed the absence of activation of extrinsic by ELISA (Table 5) assay showed significant
apoptotic pathway. absence of necrosis (~twofold lower level of
The investigation of inflammation-, apoptosis- secreted hsTnT). Secreted apoptotic biomarkers,
, necrosis-, and fibrosis-related biomarkers in similar to the EMBs, did not show activation of
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 191
Fig. 1 Evaluation of inflammation biomarkers in matory cytokines. The digital quantification of inflamma-
PVB19-negative and -positive EMBs and serums. tory infiltrates was done as described in Method part. Data
(a) Inflammatory infiltrates in EMBs. (b) Secreted inflam- are shown as means SD and are significant at p 0.05
compared to the PVB19-negative ones (Fig. 3c), of intensive necrosis in PVB19-positive DCM
which might be related to the absence of biopsies (Fig. 4a). The decreased release of high-
apoptosis in EMBs: the more intramyocardial sensitivity troponin T (hsTnT), a serum marker of
components are secreted, the less remains in the cardiomyocyte necrosis, in PVB19-positive
myocardium. patients’ serums compared to the PVB19-negative
samples confirmed the absence of necrosis in virus-
positive myocardium (Fig. 4b).
3.5 The Necrosis in PVB19-Negative
and PVB19-Positive
Myocardiums 3.6 The Correlation Analysis
of Apoptotic and Fibrotic
The level of necrosis in PVB19-positive and Biomarkers in PVB19-Positive
PVB19-negative EMB samples was investigated Myocardiums
histochemically by hematoxylin and eosin (H&E)
staining: the necrotic cells should have bigger and Since many measured biomarkers did not show
swollen nuclei compared to the healthy ones. His- the significant changes in PVB19-positive EMBs
tological staining showed only few bigger cell compared to the PVB19-negative ones, we have
nuclei in PVB19-positive EMB compared to the investigated their correlation tendencies in order
virus-negative samples and confirmed the absence to evaluate what processes could go on in the viral
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 193
Fig. 2 The histochemical evaluation of fibrosis in virus- quantification of fibrosis in PVB19-negative and
negative and -positive EMB samples. The microscopical PVB19-positive myocardium as described in method
images of Masson’s trichrome stained PVB19-negative part. Data are shown as median and interquartile range
(a) and PVB19- positive EMBs (b) representative histo- and are significant at p 0.05. (d) Correlation analysis of
logical micrographs of each group are shown. (c) Digital cardiac fibrosis and MMP1 in PVB19-positive EMBs
myocardium if no antiviral therapy would be intrinsic and extrinsic apoptotic pathways can be
applied. activated in viral myocardium (caspase-9 signifi-
The more intensive or long-lasting PVB19 cantly correlates with caspase-8) (Fig. 5b).
infection in the EMBs might activate apoptotic Finally, the inverse correlation between apo-
cell death: the extrinsic apoptotic pathway Fas ptotic DNA fragmentation and fibrosis in PVB19-
receptor significantly correlates with an executing positive EMBs (Fig. 5d) suggests that persistent
apoptotic protease caspase-3 (Fig. 5a); both presence of PVB19 in the myocardium can
194 D. Bironaitė et al.
Fig. 3 The evaluation of apoptosis by ELISA in proteins in viral EMBs. (c) The increased levels of caspase
PVB19-negativeand -positive biopsies and serums. 8, 9 and 3 in the PVB19-positive serums. Data are shown
(a) The decreased levels of caspase 8, 9, 3 in viral as median and interquartile range. Data are significant at
EMBs. (b) The decrease of mitochondria protecting p 0.05
activate cell death (DNA fragmentation) rather 3.7 The Correlation Analysis
than fibrosis (Fig. 5c) with a primary role of of Secreted Biomarkers
extrinsic apoptotic pathway in it (Fig. 5d). There in PVB-Positive Patients’ Serums
is also a possibility that further presence of
PVB19 in myocardium could sensitize it for fur- The correlation analysis of secreted biomarkers
ther toxic exposures. also confirmed the decrease of fibrosis in
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 195
Fig. 4 The level of necrosis in PVB19-negative and -pos- Representative histochemical images of each group are
itive EMBs and serums. (a) The intensity of necrosis in shown. Magnification: x10. (b) Secretion of high sensitiv-
PVB19-negative and -positive EMBs estimated histo- ity troponin T (hsTnT) in PVB19-negative and -positive
chemically (hematoxylin and eosin staining) and evaluated serums. Data are shown as median and interquartile range.
according to the swollen nuclei (blue staining). Data are significant at p 0.05
Fig. 5 The correlation analysis of apoptosis and fibrosis EMBs. (c) Significant inverse correlations between
processes in PVB19- positive vs -negative myocardium. fibrosis and apoptosis-initiated DNA fragmentation.
(a) Significant correlation of extrinsic apoptotic pathway (d) Significant correlation between MMP1 inhibitor
activator Fas receptor with executing caspase 3 in viral TIMP1 and Fas receptor in EMBs. (e) Data are significant
EMBs. (b) Significant correlations of extrinsic (caspase 8) at p 0.05
and intrinsic (caspase 9) apoptotic pathways in viral
50% of all idiopathic DCM cases, chronic virus et al. 2010; Pankuweit et al. 2003; Pankuweit
persistence was observed, which was associated et al. 2005). The initial myocardial injury or
with a worse heart functioning compared to the acute viral myocarditis usually lasts for a very
virus-negative ones (Pankuweit et al. 2000; short period subsequently activating infiltration
Tschöpe et al. 2005). It has long been thought of inflammatory cells and autoimmune response,
that the adeno- and enteroviruses are the most while DCM phase is most hardly diagnosed and
cardiotropic viruses, but later on PVB19 was least studied and is the most difficult to treat
found in up to 50% of all DCM cases and (Mason 2003; Zimmermann et al. 2010). It
associated with worse outcome (Zimmermann explains why many authors choose to explore
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 197
Fig. 6 The correlation analysis of secreted biomarkers in (b) The inverse correlation between TIMP1 and collagen
PVB19- negative and -positive serums. (a) The significant synthesis markers PICP in viral serums. Data are signifi-
correlation between TIMP1 and MMP9 in viral serums. cant at p 0.05
the more pronounced myocarditis than DCM. In mm2) (Cerqueira et al. 2002). In this study, the
addition, the timely identification of viral levels of inflammatory infiltrates in PVB19-
myocardial infection could significantly improve positive and PVB19-negative myocardiums
therapeutic efficiency of viral DCM by were similar: 10 and 11 of CD3-positive cells
preventing further myocardial remodeling. There- (T cells/mm2) and 5 and 3,5 CD68-positive cells
fore, in this study, we have investigated the ongo- (macrophages/mm2) in PVB19-positive and
ing inflammation, fibrosis, apoptosis, and PVB19-negative EMBs, respectively. The infil-
necrosis during chronic (around 3 years from the tration of T cells and/or macrophages usually
DCM diagnosis) presence of PVB19 in idiopathic results in acute or chronic inflammatory processes
DCM myocardiums and serums. The expression that impair cardiac functioning by direct cytotoxic
of PVB19 in DCM myocardiums has been effects or by the activation of other inflammatory
analyzed by qPCR, and all samples were divided cells (Blanton et al. 2019; O’Rourke et al. 2019;
into PVB19-negative and PVB19-positive groups Lafuse et al. 2020). There is also a possibility that
for the further investigation of most typical DCM more intensive infiltration of macrophages in viral
pathophysiological biomarkers and their myocardium compared to the nonviral needs
intercorrelations. more intensive stimulus than PVB19.
It has been shown that the acute viral myocar- Similar to the EMBs, PVB19 did not stimulate
ditis associates with a high level of inflammation secretion of IL-1, TNF-α, and IL-6. The decreased
caused by a nonstructural protein (NS1) of secretion of all tested pro-inflammatory cytokines
PVB19 (Duechting et al. 2008), while other stud- might be also related to the low infiltration of
ies showed that the presence of PVB19 in myo- macrophages, a known inducer of inflammation,
cardium is irrelevant to the acute myocarditis or in PVB19 myocardium (Lafuse et al. 2020;
DCM (Schenk et al. 2009). It is agreed that the Martinez et al. 2008). On the other hand, signifi-
intramyocardial inflammation needs 14 of cantly decreased level of IL-6 (a known myokine
inflammatory infiltrates (14 leucocytes/mm2 with antiapoptotic and heart protecting effects)
including up to 4 monocytes/mm2 with the pres- (Terrell et al. 2006; Fontes et al. 2015) in
ence of CD3-positive T lymphocytes 7 cells/ PVB19-positive samples could have a negative
198 D. Bironaitė et al.
impact on the myocardium. On the other hand, it The levels of other MMPs, such as MMP9 and
was shown that only chronic, not short-time, IL-6 MMP13, in the PVB19-positive vs nonviral
upregulation is associated with the worse heart DCM myocardiums did not differ significantly.
outcome (Terrell et al. 2006). It was shown that MMP9 can be located in
Many heart failure models showed fibrosis to mitochondria and macrophages and is released
be inflammation-related process (Bacmeister during the acute damage of myocardial cells
et al. 2019). The main regulator of ECM accumu- (Moshal et al. 2008; Newby 2016). The MMP9
lation and/or degradation is matrix metallopro- intramyocardial activation might also require
teinases (MMPs) family composed of more than stronger stimulus than chronic presence of
20 types of zinc-dependent proteases actively PVB19 in DCM myocardium (Lin et al. 2017).
participating in the heart tissue functioning (Lee Although the role of TIMP-1 in myocardial fibro-
2001). It has been shown that interstitial sis is not well defined, the proteolytic activity of
collagenases, such as MMP1 and MMP13, and MMPs should be kept in check by TIMPs, the
gelatinases MMP2 and MMP9 are significantly predominant inhibitors of MMPs in myocardium
expressed in mammalian myocardium and partic- (Moore et al. 2012; Yang et al. 2019). Data of this
ipate in the heart ECM degradation (Spinale study show that the level of TIMP1 in PVB19-
2002). Since the myocardium is mainly com- positive myocardiums was almost twice lower
posed of collagen type I (50–85%) and III than in PVB19-negative ones, and TIMP1 secre-
(10–45%), its degradation due to persistent pres- tion strongly correlated with the secretion of
ence of PBV19 can also have a crucial negative MMP9 and inversely with collagen I synthesis
effect on the myocardium (Weber et al. 1993; biomarker PICP. It suggests that released
Collier et al. 2012). On the other hand, the excess TIMP1 is no longer inhibiting intramyocardial
of collagen I synthesis can initiate myocardial MMP9 that could degrade intramyocardial
stiffness in hypertensive heart disease, aortic ste- collagens or even initiate apoptosis.
nosis, or late stage DCM (Díez et al. 2002; Okada The absence of intensive inflammation in
et al. 1996). In congestive heart failure mice PVB19-positive myocardiums also did not induce
model, the activation of MMP1 was shown to be neither apoptosis nor necrosis. Despite that, the
important for the left ventricle (LV) remodeling persistent presence of PVB19 in human EMBs
and heart dysfunction (Kim et al. 2000). The can impair functioning of mitochondria by strong
MMP1 was also confirmed to cleave mainly declining (~threefold) of mitochondria membrane
collagen I, while MMP2 in addition cleaves a protecting protein Bcl2 and intramitochondrial
collagen III, another important heart structural Hsp60 compared to the nonviral samples. The
protein (Steffensen et al. 1995; Patterson et al. activation of intrinsic/mitochondrial apoptotic
2001). Moreover, the MMP1 can split the colla- pathways is mainly regulated by the balance of
gen I into two fragments that are further broken antiapoptotic Bcl-2 and proapoptotic Bax family
down into smaller fragments by MMP2 and members (Elmore 2007) and needs harsh oxidative
MMP9 (Funck et al. 1997). Both MMP1 and stress or inflammation-related stimuli (Wang et al.
MMP2 also can directly remodel collagen I 2013). However, the correlation analysis of
through binding to its ά-2 chain (Gioia et al. measured apoptotic biomarkers revealed a possible
2010). In this study, the levels of MMP1 and further activation of not only extrinsic but also an
MMP2 have been significantly (by 2,2- and 2,4 intrinsic apoptotic pathway by PVB19 infection.
fold) upregulated in PVB19-positive The presence of Hsp60 in primary cardiomyocytes
myocardiums compared to the virus-negative was shown to protect mitochondria as well by
EMBs that could remodel DCM myocardium. upregulating mitochondria protecting
The significant inverse correlation between antiapoptotic Bcl-xl and Bcl-2, and reducing the
MMP1 and fibrosis also confirmed MMP1 level of proapoptotic Bax (Shan et al. 2003).
participating in myocardial ECM degradation It is well-known that cardiac tissue requires a
rather than in fibrosis. high level of energy in order to properly pump
Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human. . . 199
blood, while mitochondria are one of the key further apoptosis activation if PVB19 persists in
players in mammalian heart energy supply myocardiums. It may be that PVB19 can sensitize
(Ramaccini et al. 2021) – mitochondrial dysfunc- myocardiums to other toxic and/or viral
tion was shown to be associated with the heart infections.
diseases including DCM (Bonora et al. 2019). Finally, data show that chronic presence of a
Moreover, a number of other mitochondrial dys- PVB19 in human DCM myocardium is not a
function such as oxidative phosphorylation benign phenomenon: persistence of PVB19 in
(OXPHOS), metabolic, fatty acid, or cardiolipin DCM myocardium negatively affects ECM integ-
metabolism (Barth syndrome) correlate with the rity and energetic status of DCM myocardium.
incidents of DCM (Marin-Garcia et al. 2000; Data suggest that antiviral therapy for PVB19-
Gebert et al. 2009). The chronic presence of positive DCM patients may be applicable in
PVB19 in DCM myocardium can also sensitize order to prevent further myocardial degradation.
it to the other toxic viral infections such as coro-
navirus (Naneishvili et al. 2020). So, the data of Acknowledgments This work was supported by grants
this study suggest that chronic presence of (No. MIP-086/2012 and MIP-011/2014) of the Research
Council of Lithuania and the European Union, EU-FP7,
PVB19 in myocardium is not an innocent phe-
SarcoSi project (No. 291834).
nomenon, but can slowly degrade myocardial
ECM through the activation of MMPs and impair
Limitations The main limitation of this study, due to the
mitochondrial functioning, leading to the worse ethical problems, is the absence of healthy patients’
heart energy supply and tissue contraction. The samples. Despite this, the main goal of this study was to
antiviral therapy can be useful to prevent viral investigate the main pathophysiological processes ongoing
during chronic presence of PVB19 in DCM myocardium
DCM and further pathophysiologic heart tissue
and its impact to the further myocardium remodeling. The
remodeling. data suggest that antiviral therapy can be recommended to
treat viral DCM.
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https://doi.org/10.1007/978-3-031-02378-1
# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022
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204 Index