ORIGINAL RESEARCH
published: 03 March 2020
doi: 10.3389/fendo.2020.00086
Is Umbilical Cord Blood Therapy an
Effective Treatment for Early Lung
Injury in Growth Restriction?
Beth J. Allison 1,2*, Hannah Youn 1,2 , Atul Malhotra 1,3 , Courtney A. McDonald 1,2 ,
Margie Castillo-Melendez 1,2 , Yen Pham 1,2 , Amy E. Sutherland 1,2 , Graham Jenkin 1,2 ,
Graeme R. Polglase 1,2 and Suzanne L. Miller 1,2
1
The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Australia, 2 Department of Obstetrics and
Gynaecology and Paediatrics, Monash University, Clayton, VIC, Australia, 3 Monash Newborn, Monash Medical Centre,
Clayton, VIC, Australia
Edited by:
Richard Ivell,
University of Nottingham,
United Kingdom
Reviewed by:
Dana Manuela Savulescu,
National Institute of Communicable
Diseases (NICD), South Africa
William Colin Duncan,
University of Edinburgh,
United Kingdom
Janna L. Morrison,
University of South Australia, Australia
*Correspondence:
Beth J. Allison
beth.allison@hudson.org.au
Specialty section:
This article was submitted to
Reproduction,
a section of the journal
Frontiers in Endocrinology
Received: 15 November 2018
Accepted: 11 February 2020
Published: 03 March 2020
Citation:
Allison BJ, Youn H, Malhotra A,
McDonald CA, Castillo-Melendez M,
Pham Y, Sutherland AE, Jenkin G,
Polglase GR and Miller SL (2020) Is
Umbilical Cord Blood Therapy an
Effective Treatment for Early Lung
Injury in Growth Restriction?
Front. Endocrinol. 11:86.
doi: 10.3389/fendo.2020.00086
Fetal growth restriction (FGR) and prematurity are often co-morbidities, and both are risk
factors for lung disease. Despite advances in early delivery combined with supportive
ventilation, rates of ventilation-induced lung injury (VILI) remain high. There are currently
no protective treatments or interventions available that target lung morbidities associated
with FGR preterm infants. Stem cell therapy, such as umbilical cord blood (UCB) cell
administration, demonstrates an ability to attenuate inflammation and injury associated
with VILI in preterm appropriately grown animals. However, no studies have looked at the
effects of stem cell therapy in growth restricted newborns. We aimed to determine if UCB
treatment could attenuate acute inflammation in the first 24 h of ventilation, comparing
effects in lambs born preterm following FGR with those born preterm but appropriately
grown (AG). Placental insufficiency (FGR) was induced by single umbilical artery ligation
in twin-bearing ewes at 88 days gestation, with twins used as control (appropriately
grown, AG). Lambs were delivered preterm at ∼126 days gestation (term is 150 days) and
randomized to either immediate euthanasia (unventilated controls, AGUVC and FGRUVC )
or commenced on 24 h of gentle supportive ventilation (AGV and FGRV ) with additional
cohorts receiving UCB treatment at 1 h (AGCELLS , FGRCELLS ). Lungs were collected at
post-mortem for histological and biochemical examination. Ventilation caused lung injury
in AG lambs, as indicated by decreased septal crests and elastin density, as well as
increased inflammation. Lung injury in AG lambs was attenuated with UCB therapy.
Ventilated FGR lambs also sustained lung injury, albeit with different indices compared to
AG lambs; in FGR, ventilation reduced septal crest density, reduced alpha smooth muscle
actin density and reduced cell proliferation. UCB treatment in ventilated FGR lambs
further decreased septal crest density and increased collagen deposition, however, it
increased angiogenesis as evidenced by increased vascular endothelial growth factor
(VEGF) expression and vessel density. This is the first time that a cell therapy has
been investigated in the lungs of growth restricted animals. We show that the uterine
environment can alter the response to both secondary stress (ventilation) and therapy
(UCB). This study highlights the need for further research on the potential impact of novel
therapies on a growth restricted offspring.
Keywords: growth restriction, ventilation induced lung injury, umbilical cord blood (UCB), treatment, animal model
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INTRODUCTION
show strong anti-inflammatory benefits, and they are a feasible
postnatal treatment with low immunogenicity (25, 26, 28).
Within the lung, UCB therapy is thought to reduce inflammation
through paracrine effects. Accordingly, we used our established
model of ovine fetal growth restriction to examine (i) the
effects of preterm birth and ventilation on FGR lungs, and (ii)
if UCB could be a potential new treatment for VILI in FGR
and/or AG infants. To focus on acute inflammation and injury,
this study examined the first 24 h postnatally in FGR and AG
preterm lambs.
Fetal growth restriction (FGR) is a common complication of
pregnancy, where a fetus fails to reach its expected growth
potential, primarily due to placental insufficiency (1). FGR
significantly increases the risk of morbidity and pulmonary
conditions following preterm birth, with increased rates of
bronchopulmonary dysplasia and pulmonary hypertension
(2, 3). Despite the increased risk of pulmonary complications,
lung pathology following FGR remains contentious. We
and others have found comparable lung weight, structure,
surfactant protein expression, and ventilation requirements
compared to appropriately grown (AG) cohorts (4, 5).
However, it is evident that early and late onset FGR have
differential effects (6), and animal studies to date have
primarily induced FGR during late gestation, and it is, thus,
possible that crucial lung development has already occurred
at this stage (7); whereas preclinical studies of long term
growth restriction describe altered surfactant protein (8, 9)
disrupted alveolarization, with thickened parenchyma (10)
and large alveoli resulting in reduced alveolar and vascular
density (11).
There is currently no cure or therapy for FGR. Current
treatment of FGR primarily involves the adjustment of the
delivery time, thus infants are often delivered preterm (<37
weeks gestation), particularly those with early-onset FGR (12).
Prematurity itself is a significant risk factor for pulmonary
morbidity and necessitates medical interventions such as
mechanical ventilation. Whilst ventilation is usually essential for
survival in such scenarios, it has the potential to exacerbate
pathology in FGR lungs, particularly since lung development
may already be adversely affected by the chronic hypoxemia
caused by placental insufficiency (11). The resultant lung
injury after birth is known as ventilation induced lung injury
(VILI). VILI and elevated inflammation cause direct tissue
injury and in turn, exacerbate lung inflammation. Long term
ventilation can reduce alveoli number, disrupt vasculature
and alveolar architecture (13–16), hampering lung mechanics
and necessitating the further need for assisted ventilation.
Current treatment focuses on ensuring the survival of the FGR
infant while ameliorating the detrimental effects of FGR on
the lungs.
Umbilical cord blood (UCB) cells have been highlighted as a
potential treatment for infants born preterm, due to their potent
anti-inflammatory properties and easy access (17). Preclinical
studies using specific stem like cell populations present within
UCB have shown promising anti-inflammatory and immune
modulatory effects in VILI of preterm animals (15, 18). UCB
provides a unique source of the functionally important stem
like cells that may each play a modulatory role in preventing
injury (19). UCB therapy has been examined for prevention and
repair of brain injury (20, 21), and has also shown promise
in clinical trials where administration improved motor and
neurodevelopmental outcomes in children with cerebral palsy
(22, 23). UCB is comprised of many cell types including cells that
mediate hematopoiesis and vascular growth (24–27). UCB also
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METHODOLOGY
Umbilical Cord Blood Collection
Umbilical cord blood (UCB) was collected from separate healthy
term ovine pregnancies. UCB was collected during cesarean
section under general anesthesia. Approximately 90 mL of UCB
was collected from the umbilical veins into heparinized tubes.
UCB was diluted 1:1 with phosphate buffered saline and
centrifuged at 3,200 rpm at RT for 12 min with no brake. The
buffy coat was isolated to obtain the mononuclear cells (MNCs)
and red blood cell lysis of this fraction performed. Cells were
counted using trypan blue exclusion and a hemocytometer, and
cells were cryopreserved at ∼25 million cells/ml in freeze media
(10% DMSO, 40% FBS and 50% DMEM/F12) until required.
A minimum of three cryopreserved UCB donors was pooled
after thawing and before administration to reduce intra-sample
UCB variation.
Fetal Surgery
Aseptic surgery was performed on anesthetized (sodium
thiopentone 20 mL; Pentothal; Boehringer Ingelheim, Australia;
maintenance inhaled isoflurane 2–5%) Border-Leicester pregnant
ewes (n = 17) carrying twin-pregnancies at 88 days gestation
(term is 150 days). Prophylactic antibiotics were administered
via the maternal jugular vein, including 5 mL of Engemycin
(Engemycin 100, Coopers, MSD Animal Health, New Zealand)
and 1 g of ampicillin (Ampicyn 1 g, Mylan N.V., USA). Following
a thorough cleaning of the surgical sites, the fetus was exposed
via cesarean section. Marcain (5 mL, Marcain (0.5%) with
Adrenaline, Aspen Pharmacare Australia NSW, Australia) was
applied to all surgical sites prior to incision to provide analgesic
coverage. Single umbilical artery ligation was performed by
placing two silk ties tightly around one of the umbilical arteries,
that causes chronic placental insufficiency and fetal growth
restriction (FGR). In control twins, the umbilical cord was
handled but not ligated. The fetus was returned to the uterus
and abdominal incisions were repaired. A maternal jugular vein
catheter was inserted for antibiotic administration. Following
surgery ewes were randomly allocated to an experimental
group (UVC n = 6 ewes, V n = 5 ewes or CELLS
n = 6 ewes).
For 3 consecutive days after surgery, antibiotics [to the
fetus (Ampicillin, 1 g via the amniotic catheter] and the
ewe [Engemycin 5 mL intravenous (i.v.)] and analgesia
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UCB for FGR Lung Injury
Post-mortem
[Panadol (100 mg/mL, Apotex, NSW, Australia) suppository]
were administered.
At 24 h, ventilated lambs were euthanized with an overdose of
20 mL of phenobarbitone, whilst unventilated control groups
were immediately euthanized at 125 ± 1 days gestation via an
overdose of phenobarbitone. Lambs were weighed and lungs
isolated for collection. The left bronchus was ligated before the
left lung was removed distal to the ligature. The left lung was
snap frozen in liquid nitrogen for RNA processing. The right
whole lung was pressure fixed at 20 cmH2 O via the trachea
with formalin. Nine sections (2 cm3 ) of the lung were randomly
selected from an area devoid of major airways from each lobe
(upper, middle, lower) and processed for assessment of lung
histology. Lung sections were embedded in paraffin, then cut
into 5 µm sections and mounted on to Superfrost Plus slides for
histological and immunohistochemical analysis.
Experimental Design
The ewe and fetuses were monitored daily until 124 days
of fetal gestation. At 124 and 125 days, ewes received
11.4 mg betamethasone intramuscularly (Celestone Chronodose,
Schering Plow, Sydney, Australia). At 126 days, ewes (n = 11)
and their fetuses (n = 21) in the ventilation groups (AGV
n = 6, FGRV n = 6 and AGCELLS n = 6, FGRCELLS , n =
5) underwent an additional cesarean section or post-mortem
(AGUVC n = 6, FGRUVC n = 5). At this time, there had
been n = 2 in utero deaths in the FGR groups; n = 1 in the
FGRUVC group and n = 1 in the FGRCELLS group, hence the
reduced number in these two groups at this timepoint. Following
maternal anesthesia (sodium thiopentone 20 mL; maintenance
inhaled isoflurane 2–5%), the first lamb was exteriorized and
intubated with an endotracheal tube (size 4.0 mm). Lung liquid
was drained passively and a transcutaneous arterial oxygen
saturation (SpO2 ) probe (Masimo, Radical 4, CA, USA) was
placed around the right forelimb of the lamb and the output
digitally recorded.
The umbilical cord was then clamped and cut, the lambs
were delivered, dried, weighed and placed on an infant warmer
(Fisher and Paykel Healthcare, Auckland, New Zealand) for
initiation of assisted ventilation. An umbilical vein and artery
were immediately catheterized for maintenance of anesthesia
and analgesia (Alfaxane i.v. 5 mg/kg/h; Jurox, East Tamaki,
Auckland, New Zealand). Arterial pressure was digitally recorded
in real-time (1 kHz, Powerlab; ADInstruments, Castle Hill, NSW,
Australia). The lambs were anesthetized for the entirety of the
experiment to prevent spontaneous breathing. Ventilation was
commenced using positive pressure ventilation with PIP set at
30 cmH2 O and PEEP at 5 cmH2 O (Babylog 8000+, Dräger,
Lübeck, Germany): inspiratory time was 0.4 s and expiratory time
was 0.6 s. Lambs were ventilated with warmed, humidified gas
with an initial fraction of inspired oxygen (Fi O2 ) of 0.4 and
subsequently adjusted to maintain SaO2 between 90 and 95%.
At 10 min, all lambs received surfactant (Curosurf, 100 mg/kg,
Chiesi Farmaceutica, Italy). At 20 min, ventilation continued in
volume guarantee mode set at 5–7 ml/kg, which is the tidal
volume for lambs at this gestation (29). Physiological parameters
pH and PaCO2 were kept within normal limits (7.2–7.4 and
45–55 mmHg, respectively) by adjusting the ventilator rate and
inspired O2 levels. Lamb well-being was monitored throughout
ventilation via assessment of the partial pressure of arterial
oxygen (PaO2 ) and carbon dioxide (PaCO2 ), oxygen saturation
(SaO2 ), pH, hematocrit, glucose and lacate with regular blood gas
samples (ABL 700 blood gas analyzer; Radiometer, Copenhagen,
Denmark). Lambs were ventilated for 24 h.
For groups that received UCB (AGCELLS n = 6 and
FGRCELLS n = 5), 25 million cells/kg was administered
intravenously to lambs at 1 h after birth, control groups (AGV
n = 5 and FGRV n = 5) were administered the equivalent
volume of saline. UCB cells were quantified via cell counts
of UCB mononuclear cells to establish an accurate dose prior
to administration.
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Detecting Stem Cell Migration
To detect if UCB stem cells were present in the lungs of
treated lambs, the UCB cells were tagged with carboxyfluorescein
succinimidyl ester (CFSE) before administration (30). Cut
lung sections were dewaxed and counter-stained in Hoechst
(Invitrogen, USA) and coverslipped. Stem cell identification
was conducted using fluorescent microscopy (Olympus BX41, Japan).
Histological Analysis of Lung Morphology
Gross histological pathology and parenchymal elastin was
detected via Hematoxylin and Eosin and Hart’s elastin stains,
respectively, as previously described (31) Masson’s Trichrome
was used to identify collagen fibers (32). Three sections
(obtained as described above) were randomly selected for
each histological assessment. Quantification of histology is
outlined below.
Immunohistochemistry
Lung tissue was immunostained for Ki67, α-smooth
muscle actin and CD45. Ki67 and α-smooth muscle actin
immunostaining was carried out as previously described
[(5, 33) see Supplementary Table 1]. For immunostaining of
CD45, slides were heated in a 60◦ C oven for 2 h to remove
excess wax, followed with histolene clearing and ethanol
rehydrating steps. Antigen retrieval was performed by boiling
tissue sections in 0.01 M Citrate buffer (pH 6.0) for 3 ×
10 min bursts. Sections were then washed in phosphate buffer
solution (PBS) before endogenous peroxidase in the tissue was
blocked with 3% hydrogen peroxide for 10 min. Tissue sections
were washed and then slides were blocked in Serum-Free
Protein Block (DAKO) before incubation with the primary
antibody CD45 (BD Pharmingen Rat Anti-Mouse, 1:500) for
60 min. Sections were washed in PBS and then incubated with
biotinylated secondary antibody (Rabbit anti-mouse, 1:200)
for 60 min followed with streptavidin horseradish peroxidase
and developed with diaminobenzidine (DAB) and hydrogen
peroxide. Sections were counterstained with hematoxylin and
dehydrated with ethanol and histolene before mounting with a
coverslip. All immunostains were performed in the presence of a
negative control.
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TABLE 1 | Total sample size, animal and lung weights and sex of fetuses and lambs.
AGUVC
n
FGRUVC
AGV
FGRV
AGCELLS
FGRCELLS
6
5
6
6
6
5
Animal weight (kg)
3.56 ± 0.13
2.25 ± 0.17#
3.21 ± 0.17
2.46 ± 0.22#
3.40 ± 0.18
2.29 ± 0.17#
Lung corrected for body weight (g/kg)
35.94 ± 1.98
31.04 ± 3.07
29.33 ± 0.91
30.36 ± 3.22
27.32 ± 1.55
29.44 ± 1.90
5 (83)
2 (40)
2 (33.3)
2 (33.3)
5 (83)
2 (40)
Males, n (%)
AG, appropriately grown; UVC, unventilated control; FGR, growth restricted; V, ventilated; CELLS, animals treated with umbilical cord blood cells. # Indicates p < 0.05 for growth effects
using a 2-way ANOVA.
Cytokine Array
To assess cytokine expression in the lungs, frozen lung tissue
was weighed out in 50–100 mg quantities, for protein expression
of pro-inflammatory and anti-inflammatory cytokines. The
concentrations of interferon gamma (IFNγ ), interleukin (IL)-17A,
IL-21, IL-8, IL-10 TNFα, and VEGF-A in lung tissue lysate were
measured using an ovine cytokine array (ovine QAO-CYT-1-1,
RayBiotech, Georgia, USA).
Analysis
For histological and immunohistochemistry analysis, five
random fields of view were taken of each section and analyzed by
a single blinded observer (H.Y.). Images were non-overlapping
and excluded large airways or vessels.
Lung morphology was assessed through quantification of
tissue to airspace ratio and density of secondary septal crests
as previously described (4). Elastin, collagen and αSMA density
were assessed through Smart Segmentation on Image Pro
Premier (Media Cybernetics, USA) (16). Elastin and collagen
were then expressed as ratios of lung tissue. Manual point
counting was utilized to assess Ki67 and CD45 to tissue ratios
(16). Measurement of vascular vessel number was assessed using
αSMA immunostained tissue by a single observer blinded to the
experimental group (B.J.A.). Vessels were identified by positive
staining and were only included when a full cross section of the
vessel was visible in the field of view.
Data are expressed as mean ± standard error of the mean
(SEM). Statistical analysis was performed with SPSS using
a mixed model using growth and treatment as factors in
all immunohistochemical and morphological assessments and
growth, treatment and time in ventilation parameters. Where
significant interactions were detected, differences were isolated
with post-hoc Tukey’s testing. Statistical significance was accepted
as P < 0.05.
FIGURE 1 | Ventilation parameters. Mean ± SEM tidal volume VT (mLs.kg−1 )
and peak inspiratory pressure (PIP) in appropriately grown (AGV , white circles,
n = 5), growth restricted (FGRV , black circles n = 5) and appropriately grown
and growth restricted treated with umbilical cord blood cells (AGCELLS , white
squares n = 6; FGRCELLS , black squares n = 5) over the experimental period
(hours) *indicates significant differences (p < 0.05) across time.
32% <AGCELLS (P = 0.01, Table 1). Lung weight, corrected for
body weight, was not different across groups.
Ventilation Parameters
There were no differences in tidal volumes (VT , 5–6 mL/kg)
between groups. Peak inspiratory pressure (PIP) required to
achieve VT was initially not different between groups, however
PIPs were significantly (P > 0.003) increased at 20 h in FGRV
lambs compared to all groups (Figure 1). There was no effect of
FGR or UCB on the requirement for PIP. Lung compliance was
not different between groups (data not shown).
RESULTS
Lamb Characteristics
Lamb characteristics are presented in Table 1. Single umbilical
artery ligation (SUAL) resulted in ∼30% overall reduction in
birth weight in FGR lambs. SUAL also resulted in the death of two
fetuses, one in the UVC and one in the CELLS group. FGRUVC
weighed 37% <AGUVC (P = 0.0001), whilst FGRV weights were
23% lower than AGV (P = 0.04) and FGRCELLS lambs weighed
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Stem Cell Migration
Lung tissue was examined for the presence of CFSE tagged UCB
cells in all ventilated groups (Figure 2). Fluorescing cells were
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FIGURE 2 | Representative lung morphology images. Photomicrographs of Masson Trichrome stained sections in appropriately grown (A AGUVC , C AGVENT , E
AGCELLS ) and growth restricted (B FGRUVC , D FGRVENT , F FGRCELLS ) animals. Fluorescent tagged cell in lung parenchyma UCB cells present within parenchymal lung
tissue (blue) and a fluorescing UCB cell (green). Magnification ×400 (Gi and at higher magnification Gii).
size for morphological and immunohistochemical analysis is
AGUVC n = 6, AGV n = 6, AGCELLS n = 6, FGRUVC n = 4, FGRV
n = 6 and FGRCELLS n = 4.
Tissue to airspace ratio was not altered by FGR or ventilation
(Figure 3A). Heterogeneous lung injury was observed in AGV
and FGRV compared to their unventilated cohorts, with areas
containing thickened blood-air barriers, contrasting against
apparent in all cell treated animals, and were not present in saline
controls (Figure 2G).
Lung Morphology
Two lungs were not appropriately fixed (one from FGRUVC
and one from FGRCELLS ) and were thus excluded from
morphological and immunohistochemical analysis. Final sample
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FIGURE 3 | Lung parenchymal and vascular structure. Data are mean ± SEM tissue airspace ratio (A), secondary crest density (B), arteriolar vessel wall number (C)
and collagen (D) elastin (E) and α-smooth muscle actin (F) density (corrected for total tissue area) in appropriately grown (AG) and growth restricted (FGR)
unventilated controls (AGUVC and FGRUVC , white circles), following ventilation (AGVENT and FGRVENT , black circles) and following ventilation and cell treatment (AGCELLS
and FGRCELLS, gray circles). Data were compared using a two-way ANOVA. *Indicates p < 0.05 treatment effects and # indicates p < 0.05 growth effects using a
two-way ANOVA.
in vessel density were observed across groups in the
AG lambs.
Collagen density was not altered in either AG or FGR
lambs following ventilation (Figure 3D). Treatment with UCB
significantly increased collagen density in FGRCELLS animals
compared to FGRV and AGCELLS lambs (FGRCELLS 10.7 ± 1.4%
vs. AGCELLS 5.3 ± 0.8% and FGRV 4.5 ± 0.9%; p > 0.02).
Elastin density was significantly reduced in ventilated AG
lambs compared to AG control lambs (AGUVC 22.1 ± 2.2%
vs. AGV 13.6 ± 1.7%, P < 0.05). Elastin density was restored
following treatment with UCB in AG lambs (Figure 3E). Elastin
density was not different in FGR lambs either with ventilation or
UCB treatment.
We assessed positively stained α-smooth muscle actin tissue
to determine density (Figure 3F). Ventilation of FGR lambs
other regions showing prominent airway enlargement. This
heterogeneity was reduced with UCB (AGCELLS and FGRCELLS ),
although alveoli remained enlarged (Figure 2). Septal crest
density was significantly reduced following ventilation compared
to unventilated controls (Figure 3B) resulting in a 57.6%
reduction in AG lambs (AGUVC 4.2 ± 0.5 vs. AGV 2.0 ± 0.4, P
= 0.0001) and 44.6% reduction in FGR lambs (FGRUVC 5.3 ± 0.7
vs. FGRV 2.5 ± 0.5, P = 0.002, Figure 3B). Treatment with UCB
restored septal crest density in AG (AGCELLS 3.3 ± 0.2, P = 0.02)
but not FGR lambs.
Vessel number, as assessed in α-smooth muscle actinstained lungs (Figure 3C). was significantly increased in
ventilated growth restricted lambs treated with UCB compared
to unventilated, growth restricted lambs (FGRCELLS 11.2
± 1.6 vs. FGRUVC 6.8 ± 0.5, P = 0.04). No differences
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FIGURE 5 | Inflammation. Data are mean ± SEM CD45 (inflammation marker)
positive cells in appropriately grown (AG, white) and growth restricted (FGR,
black) unventilated controls (AGUVC and FGRUVC ), following ventilation (AGVENT
and FGRVENT ) and following ventilation and cell treatment (AGCELLS and
FGRCELLS ). Data were compared using a two-way ANOVA. *Indicates p < 0.05
treatment effects using a two-way ANOVA.
FIGURE 4 | Cell proliferation. Data are mean ± SEM Ki67 (cell proliferation
marker) positive cells in appropriately grown (AG, white) and growth restricted
(FGR, black) unventilated controls (AGUVC and FGRUVC ), following ventilation
(AGVENT and FGRVENT ) and following ventilation and cell treatment (AGCELLS
and FGRCELLS ). Data were compared using a two-way ANOVA. # Indicates p
< 0.05 treatment effects using a two-way ANOVA.
significantly reduced α-smooth muscle actin density (FGRV 6.8
± 1.3% vs. FGRUVC 17.3 ± 2.1%). Treatment with UCB reversed
this finding (FGRV 6.8 ± 1.3% vs. FGRCELLS 18.6 ± 4.6%). αsmooth muscle actin density was not different in AG lambs either
with ventilation or UCB treatment.
and FGRV (P = 0.04) lambs. However, treatment with UCB
cells significantly increase VEGF protein levels in FGR lambs
compared to AG lambs treated with UCB cells (FGRCELLS
16.6 ± 0.7 vs. AGCELLS 9.9 ± 1.8, P = 0.002). There was no
difference in TNFα, IL-17A, IL-10 or IFNγ levels between groups
(Figures 6B–D,F, respectively).
Cell Proliferation
Cell proliferation was assessed in lung parenchyma using the
proliferation marker Ki67. Cell proliferation was significantly
decreased (P < 0.0001, Figure 4) in FGR, compared to AG
groups. Cell proliferation was significantly reduced by 80% in
FGRVENT compared to AGVENT , and also decreased in FGRCELLS
compared to AGCELLS (by 90%).
DISCUSSION
Postnatally, FGR infants have increased risk of lung injury
and bronchopulmonary dysplasia (BPD). Stem cell therapy has
proven benefits to reduce VILI in preterm infants (34) as well
as in reducing BPD incidence in preterm humans (33) and
in animal models of neonatal lung injury (35). However, no
previous studies have investigated if stem cell therapy is also
beneficial for very low birthweight infants affected by growth
restriction. In the current model, UCB therapy attenuated injury
in AG but not FGR lambs. Our findings in appropriately grown
lambs confirm previous studies showing improved lung structure
following administration of placental stem-like cells, such as
human amnion epithelial cells (34). Therefore, our current study
increases the body of evidence for the use of UCB as an effective
therapy for VILI in preterm infants who are appropriately
grown. UCB treatment in FGR lambs increased pulmonary
vascularization, but did not improve structural deficits in
secondary septal crests, and increased collagen deposition, which
is an early marker of fibrosis. Our research demonstrates
that after 24 h of ventilation, UCB therapy shows differential
effects in appropriately grown and growth restricted lambs,
where protection from VILI was evident in AG lambs but not
FGR lambs.
Inflammation
We used immunohistochemical analysis of CD45 to visually
examine the infiltration of inflammatory cells into pulmonary
tissue. Ventilation induced an inflammatory response in AG
lambs (AGUVC 3.9 ±.5 cells vs. AGV 16.7 ± 5.5 cells, P = 0.0079).
Inflammatory cell infiltration of the lungs was attenuated by
treatment with UCB (Figure 5, p < 0.05) in AG lambs. Neither
ventilation nor cell treatment induced a significant inflammatory
response within the lungs of FGR lambs (FGRUVC vs. FGRV p =
0.5; FGRV vs. FGRCELLS p = 0.9).
A cytokine array was performed to further characterize the
inflammatory profile within the lungs. Pro-inflammatory marker
IL-8 was significantly increased in response to ventilation, in both
AG and FGR lambs (P < 0.05), while treatment with UCB did
not reduce IL-8 levels (Figure 6A). UCB treatment significantly
increased IL-21 levels in FGR and AG lambs (Figure 6E). VEGF
concentration was significantly increased in FGR lambs treated
with UCB compared to both FGRUVC (P = 0.007, Figure 6E)
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FIGURE 6 | Inflammatory proteins. Data are mean ± SEM interleukin (IL)-8 (A), tumor necrosis factor alpha (TNFα) (B) , IL-17A (C), IL-10 (D), IL-21 (E) interferon
gamma (IFN) (F), and vascular endothelial growth factor (VEGF) (G), cytokine levels in appropriately grown (AG, white) and growth restricted (FGR, black) unventilated
controls (AGUVC and FGRUVC ), following ventilation (AGVENT and FGRVENT ) and following ventilation and cell treatment (AGCELLS and FGRCELLS ). *Indicates p < 0.05
treatment effects using a two-way ANOVA.
In the current study, we found little difference in the baseline
lung morphology between the preterm unventilated FGR and AG
fetuses, in line with previous observations from our group (4),
although we induced early-onset placental insufficiency and FGR
in this study, where we have previously examined late-onset (36).
We hypothesized that longer exposure to placental insufficiency
over a period of critical lung development would lead to the arrest
of alveolar development as observed in other preclinical FGR
studies (10, 11, 37). The latter being a probable mechanism for the
increased risk of BPD (3) in this cohort. However, we did not see
detectable differences in lung weights, when corrected for body
weight, or baseline lung morphology in this study. Differences
between the mode of inducing FGR and timing of compromise
are most likely to contribute to differences in experimental
outcomes. It is interesting to note that, despite a lack of gross
or microscopic changes in lung morphology before ventilation,
critical differences in response to ventilation were evident in
Frontiers in Endocrinology | www.frontiersin.org
the current study between AG and FGR lambs, suggesting subclinical alterations in lung development and/or biochemistry.
We have previously shown that AG and FGR newborns do
not have significantly different ventilator requirements in the
first 2 h of life (4, 38), however, in this study we extended these
findings to show that, with a prolonged period of ventilation,
FGR lambs begin to require a greater PIP to maintain VT ,
suggesting stiffer and less compliant lungs, a change that was subclinical throughout our experiment period as shown by dynamic
compliance. All our lambs received antenatal betamethasone,
which enhances surfactant production (39) and pulmonary
function (40), and temporarily preserves lung compliance (41,
42) however, this may have had decreased efficacy in FGR. The
rise in PIP over time may be a precursor to worsening VILI, lung
compliance and ventilatory requirements, and certainly suggests
that a longer period (>24 h) of study is necessary to tease apart
differences associated with FGR.
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in neonatal sheep and mice induces an upregulation of elastin
production, but not the regulators of elastin assembly, leading
to disordered accumulation of elastin along the alveolar walls
(52). The qualitative changes we observed are likely a precursor
to abnormal elastin deposition, highlighting the importance of
treating in this acute period, before structural changes. Collagen,
elastin and α-smooth muscle actin are essential structural
components of the lung (53), and perturbations to the density
and distribution of these factors will alter the mechanics of the
lung. Twenty-four hours of ventilation in our preterm lambs
decreased α-smooth muscle actin density in the FGR cohort
compared to unventilated controls. Long-term (1 month) of
ventilation increases α-smooth muscle actin (54), and thus we
may have observed a transient decrease in this study, before a
secondary compensatory increase. The mechanisms underlying
the decreased α-smooth muscle actin in this current study
are unknown, however, it is interesting to speculate on the
possible role of nitric oxide (NO). In culture, increased NO
reduces smooth muscle production, whilst inhibition of NO
results in smooth muscle accumulation (55). It is well-accepted
that growth restriction impairs NO handling (56, 57), and
we have shown decreased content and altered distribution of
the NO precursor, eNOS, following 2 h of ventilation (58).
However, exposure to hyperoxia in the first day of life may
increase local NO production due to impaired NO handling
in FGR newborns, thereby resulting in inhibition of smooth
muscle production.
Septal crest density is vital for increasing the surface area
available for gas exchange. Ventilation induced a decrease in
septal crest density in AG and FGR lambs, which is representative
of simplification of the airways, and this is a hallmark of
bronchopulmonary dysplasia (59). UCB was protective for septal
crest density in the lungs of AG lambs, but not the FGR
lambs. Despite this, we observed an improvement of injury
heterogeneity in both AG and FGR lambs with treatment.
Therefore, UCB may also promote, via paracrine mechanisms,
surfactant production to reduce atelectasis.
Strikingly, there was an increase in the collagen to tissue
ratio after UCB administration in FGR lambs. In previous
studies, UCB-derived mesenchymal stem cells (MSCs) have
increased fibroblast formation compared to those sourced from
adipose tissue or bone marrow (13, 60). Despite this, no
previous studies observed increased collagen; and UCB-MSCs
or mononuclear cells administered to mice with VILI did
not alter levels of TGF-β, a regulator of collagen production,
or collagen content 14 days after cell administration (15,
50). However, to the authors’ knowledge, there are no other
studies specifically aimed at determining the efficacy of cell
treatment in a growth restricted population. Whilst it is
possible that the increase in collagen in FGR lambs in the
current study is transient, given the relationship between
collagen deposition and fibrotic disease, this relationship requires
additional research.
Alveolar epithelial cells are a key source of increased cell
proliferation following ventilation induced lung injury (61).
Interestingly, cell proliferation was significantly increased in
ventilated AG lambs but reduced in FGR ventilated lambs. FGR
Mechanical trauma as occurs with assisted ventilation induces
an acute inflammatory response that initiates the inflammatory
cascade and stimulation of inflammatory cytokine production
(14, 16, 35). This was confirmed in the current study with
ventilation significantly increasing pro-inflammatory cytokine
IL-8 in FGR and AG lambs. In keeping with pulmonary IL8 upregulation, infiltration of immune cells into the lungs
(as evident by CD45+ expression) was also increased with
ventilation, albeit this was statistically significant only in the
AG lambs. Upregulation of inflammation following lung injury
is well-described (4, 14, 16, 43, 44). Interestingly, IFNγ and
TNFα were not altered in ventilated FGR or AG lambs. IFNγ
is recognized as a key pro-inflammatory cytokine that has
previously been shown to be up-regulated in response to lung
injury (45). We did not see an up-regulation of IFNγ in
the current study, this is likely due to the timing of lung
collection, given that we measured inflammatory proteins in
the lungs collected after 24 h of ventilation. IFNγ is seen to
increase transiently in response to the initiation of ventilation
with levels reducing over a period of hours-to-days after initial
increase (46). TNFα is also found to be released in response
to VILI in preterm neonates (45) however, pre-treatment with
betamethasone, as was given in the current study, can prevent an
increase in TNFα (47). UCB treatment in AG lambs attenuated
immune cell infiltration into lungs but did not prevent the
increase in IL-8 in either AG or FGR lambs. Moreover, UCB
induced a 1.6-fold increase in lung IL-21 in AG and FGR
lambs. IL-21 is a pro-inflammatory cytokine that promotes M2
“repair” to M1 “classically activated” macrophage phenotype,
as well as increasing CD4+ and CD8+ T cell production
(48), thus promoting inflammation. Persistence of inflammatory
markers upregulated in response to mechanical ventilation in
the current study are in contrast to previous reports where
administration of placental stem cells increased expression
of anti-inflammatory cytokines and reduced markers of lung
inflammation following hyperoxic injury, thereby preventing
downstream fibrosis and normalizing lung morphology (49).
Differences between the findings here and those of previous
studies for cell efficacy may reflect differences in the timing
of tissue collection, mode of lung injury, the stage of lung
development, or indeed the cells administered. It is perhaps
too early to speculate whether the large increase in pulmonary
IL-21 in response to UCB cells is a reparative or damaging
effect, but it is increasingly well-understood that stem cells can
modify a reparative response via immunomodulatory actions.
This, however, is contingent on the inflammatory environment at
the time of cell administration; stem cells introduced into a highly
inflammatory host inhibit the protective capacity of stem cells
(50), and can, in some instances, result in stem cells themselves
becoming pro-inflammatory (51). This is a research area that
requires further characterization, particularly for the vulnerable
fragile preterm lungs.
Consistent with previous findings, we observed suppression
of septal crests density following ventilation (16), and elastin
distribution became diffuse along the alveolar wall; in AG lambs
elastin density was significantly reduced and a similar (nonsignificant) trend was seen in lungs from FGR lambs. Ventilation
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UCB for FGR Lung Injury
the response of the organs to additional insults. It is likely
that the lung is similarly affected, where an altered response
to injury in FGR as compared to AG offspring has been
demonstrated in animal models (70) and humans (3). It follows
that treatments also may have different therapeutic ranges in
infants following a sub-optimal pregnancy, and therefore further
research is required to determine how to best target therapies to
this population.
is linked with lower levels of growth factors and decreased
pulmonary cell growth in culture (11). We have previously
shown that glucocorticoids reduce cell proliferation, both in
AG and FGR fetuses (38) but since all groups received
betamethasone, this is unlikely to have caused the difference
observed here. It is more likely that the growth restricted
lung does not respond to stretch by inducing proliferation,
a well-established mechanism in the lungs of AG infants.
Indeed, another key stretch response, the baroreflex response,
is significantly attenuated in growth restricted fetuses (62),
suggesting a possible decreased responsivity to this critical form
of stimulus. Overall, these unexpected findings re-emphasize
that even though ventilator requirements and fetal histology was
not different between groups, FGR lungs respond differently
to ventilation compared to AG lungs, and these changes may
underpin the increased vulnerability to injury and long-term
morbidity in FGR offspring. UCB treatment did not improve
cell proliferation in FGR lungs. The underlying physiology is
unknown, however investigating which cells are proliferative in
AG would be of interest.
Treatment with UCB in FGR ventilated lambs promoted
blood vessel growth as evidenced by the increased VEGF and
vessel number, a finding not seen in AG lambs. VEGF is a
potent inducer of angiogenesis and decreased VEGF expression
is seen in newborns with BPD. In vitro, both MSCs (63)
and endothelial progenitor cells (64) promote angiogenesis, via
increases in VEGF. It is possible that, given time, increased
vascularization of the lung would promote restoration of
alveolarization, given the known positive relationship between
these two factors (65). It is interesting, but not immediately
apparent, why VEGF was increased in FGR, but not AG
ventilated lambs. It is known that hypoxia increases VEGF
production, and placental insufficiency directly exposes the
developing fetus to chronic hypoxemia, which may in turn,
result in impaired or altered hypoxia sensing and handling,
and response.
How stem cells exert a reparative benefit is still not fully
understood, however several mechanisms are possible. They may
migrate to areas of injury and release trophic factors to reduce
inflammation and promote endogenous repair mechanisms or
they may alter systemic immune-modulatory responses (66).
We observed only small numbers of UCB cells within the
lungs, suggesting that their main effect was not via cell
engraftment, but rather a paracrine effect as expected. In
vitro, MSCs demonstrated preferential migration to hyperoxiainjured lung tissue rather than control medium or healthy
lung tissue (49), suggesting stem cells are specifically recruited
to sites of injury. Intra-tracheal UCB administration provides
direct access to the lung and may improve lung outcomes
(35), however, given that intubation is increasingly infrequent
in pediatrics (67), systemic administration of cells is more
clinically relevant.
It is now well-established that a poor uterine environment
has the potential to program disease in later life (68). Emerging
evidence also suggests that subtle, sub-clinical alterations are
present in the lung (69) at the time of birth, which not
only changes the function of the organ but can also alter
Frontiers in Endocrinology | www.frontiersin.org
Limitations
We administered the UCB cells at a dose of 25 million cells/kg,
based on evidence that this level is neuroprotective, and gave
this dose 1 h after birth. It is possible that this cell dose
and timing is not optimal for treatment of the lungs of the
FGR infant and highlights the need to consider the FGR
population independent of AG preterm infants. As with many
other therapeutics, the timing of stem cell administration is
vital, and it is possible that delaying cell administration until
after the primary inflammatory phase is more protective in
the lung, as has been observed in vitro (51). Further studies
should examine this possibility and also determine the effects
of UCB on VILI, in AG and FGR animals, beyond the 24h. This will offer a better understanding of how UCB may
benefit functional and morphological outcomes and chronic lung
disease. Finally, the FGR infant has multi-organ dysfunctions
and, while we only examined lung outcomes in the current study,
optimizing postnatal therapeutics for multiple organs, including
the lung, brain and cardiovascular system, must be considered.
Treatment strategies to improve neurological structure and
function are now being examined, including cell therapies. The
current study suggests that we need additional targeted research
to determine the interaction between organ systems, possible
developmental programming and postnatal treatments, such as
cell therapies.
Conclusion
FGR newborns have an increased risk of bronchopulmonary
dysplasia and, whilst there is currently no cure for FGR or lung
disease in this vulnerable cohort, UCB stem cells have shown
potential therapeutic benefits in preterm infants. Here we sought
to determine if UCB cells would also be beneficial for growth
restricted preterm newborns. Our results have demonstrated that
UCB shows promising anti-inflammatory benefits for treating
ventilation induced lung injury in appropriately grown newborn
lambs. However, UCB treatment was not equally effective for
FGR infants, where it promoted angiogenesis, did not reverse
detrimental changes in lung structure, and increased collagen
and its precursor αSMA, which may be injurious. Interestingly,
the pulmonary response to cell administration was differentially
regulated in AG and FGR lambs, wherein UCB increased
VEGF and decreased cell proliferation in FGR lambs only,
however, whether this would be beneficial or not for the
future of the offspring is yet to be determined. Our study is
the first to highlight that the FGR infant responds differently
to cell therapy, and these results suggest that developmental
programming in utero needs to be considered when giving
postnatal treatments.
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DATA AVAILABILITY STATEMENT
FUNDING
All datasets generated for this study are included in
article/Supplementary Material.
This work was supported by NHMRC Project Grant APP1083520
and the Victorian Government’s Operational Infrastructure
Support Program.
ETHICS STATEMENT
ACKNOWLEDGMENTS
Ethical approval for all experimental procedures utilized in this
project was granted through the Monash Medical Centre Animal
Ethics Committee (approval number MMCA2014-04).
We would like to acknowledge the assistance of Ilias Nitsos,
Tamara Yawno, and Michael Fahey who assisted with the
animal studies.
AUTHOR CONTRIBUTIONS
SUPPLEMENTARY MATERIAL
BA, AM, GP, and SM conceived and designed the analysis. BA,
HY, AM, CM, MC-M, YP, AS, GJ, GP, and SM collected and
contributed to the data. BA and HY performed the analysis
and BA, HY, AM, CM, MC-M, YP, AS, GJ, GP, and SM wrote
the paper.
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fendo.
2020.00086/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Allison, Youn, Malhotra, McDonald, Castillo-Melendez, Pham,
Sutherland, Jenkin, Polglase and Miller. This is an open-access article distributed
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