Biotechnology and Genetic Engineering Reviews

ISSN: 0264-8725 (Print) 2046-5556 (Online) Journal homepage: https://www.tandfonline.com/loi/tbgr20

Metabolic status of pluripotent cells and

exploitation for growth in stirred suspension
bioreactors

Brad Day & Derrick E. Rancourt

To cite this article: Brad Day & Derrick E. Rancourt (2013) Metabolic status of pluripotent cells and
exploitation for growth in stirred suspension bioreactors, Biotechnology and Genetic Engineering
Reviews, 29:1, 24-30, DOI: 10.1080/02648725.2013.801233

To link to this article: https://doi.org/10.1080/02648725.2013.801233

Published online: 24 Jul 2013.

Submit your article to this journal

Article views: 424

View related articles

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tbgr20
Biotechnology and Genetic Engineering Reviews, 2013
Vol. 29, No. 1, 24–30, http://dx.doi.org/10.1080/02648725.2013.801233

Metabolic status of pluripotent cells and exploitation for growth in

stirred suspension bioreactors
Brad Day and Derrick E. Rancourt*

Department of Biochemistry and Molecular Biology, Faculty of Medical Science, University of

Calgary, Calgary, Alberta, Canada
(Received 28 September 2012; accepted 15 February 2013)

Pluripotent stem cells are of great interest in the field of regenerative medicine.
Recent studies have shown that they maintain a glycolytic metabolic status while plu-
ripotent and wholesale changes to mitochondrial and metabolic profile occur during
differentiation. This article reviews the process and how this may be exploited in a
stirred suspension bioreactor for rapid growth while maintaining pluripotency.
Keywords: pluripotency; metabolic status; bioreactor

Introduction
Pluripotent stem cells (PSCs) possess the ability for infinite self-renewal and the capac-
ity to differentiate into any tissue within the body. PSCs typically come from two
sources, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs
are derived from the inner cell mass of the blastocyst (Evans and Kaufman, 1981).
iPSCs are formed by reprogramming a non-pluripotent somatic cell with reprogramming
factors, forcing it to take on a pluripotent state (Takahashi and Yamanaka, 2006). PSCs
can be used as a model to study early molecular and cellular process in development.
The ability to differentiate into any tissue within the body, specifically those that do not
naturally regenerate, has also spearheaded study in the field of regenerative medicine.
These cells present an alternative treatment method for diseases that at present lack
long-term solutions. Unfortunately, before advanced therapeutic techniques can be
developed, technical issues in the expansion of PSCs need to be addressed. Specifically,
the static culture system now used does not efficiently produce clinically usable cell
numbers; the system lacks culture control and culture heterogeneity exists leading to
batch variability. This is particularly true with iPSCs. To overcome this, the use of bio-
reactors is becoming more prevalent to grow both ESCs and iPSCs. In bioreactors, key
parameters such as oxygen and carbon dioxide levels can be better monitored – allow-
ing for more control over cell expansion. The bioreactor also allows for rapid expansion
while maintaining pluripotency. It is unknown what environmental factors in the
bioreactor contribute to the maintenance of pluripotency.
Recently, researchers have reported that PSCs have distinct metabolic signatures and
use different metabolic pathways compared with somatic differentiated cells. Pathway
utilization and oxygen consumption change as the cell changes from a pluripotent state

*Corresponding author. Email: rancourt@ucalgary.ca

Ó 2013 Taylor & Francis

 Biotechnology and Genetic Engineering Reviews 25

to a differentiated state. In this review, we discuss recent advances in the understanding

of the metabolic and mitochondrial differences of pluripotent regulation and how this
pertains to growth and expansion of cells within stirred suspension bioreactors.

Regulation of pluripotency
PSCs rely on molecular and environmental factors to control the balance between
pluripotency and differentiation. In the body, PSCs are found within a specific
microenvironment or niche, which helps to regulate cell fate. During development, vari-
ous factors act on PSCs to alter gene expression and induce differentiation. Interactions
between stem cells as well as interactions with adhesion molecules, extracellular matrix
and oxygen tension are all important factors in the regulation of cell niche and mainte-
nance of pluripotency (Kurosawa, Kimura, Noda, & Amano, 2006; Naveiras and Daley,
2006).
Early in differentiation the cell undergoes global gene expression changes, altering
cellular signalling and function. Genes responsible for the maintenance of pluripotency
are downregulated, while genes responsible for lineage specific differentiation are con-
versely upregulated. While the exact mechanism of maintenance is unknown, transcrip-
tional factors such as Oct4, Sox2 and Nanog play a critical role in the regulation of
pluripotency. The study of various conditions of the ESC niche is vital for the replica-
tion of in vitro cell growth. In vitro cell growth is necessary for regenerative therapies,
as cell proliferation and differentiation is controlled in stir-flasks or plates before being
introduced into the patient for therapy.
PSCs grown in vitro require the addition of leukemia inhibitory factor (LIF) in
mouse and basic fibroblast growth factor (bFGF) in human (Williams et al., 1988;
Schuldiner, Yanuka, Itskovitz-Eldor, Melton, & Benvenisty, 2000). The presence of LIF
promotes Sox2 by activating the JAK/STAT pathway and Nanog production through the
PI3K/AKT signalling cascade (Niwa, Ogawa, Shimosato, & Adachi, 2009). The exact
mechanism of bFGF is currently unknown. Both cell types are often grown on a feeder
layer of cells, typically mouse embryonic fibroblasts (MEFs). These cells are supportive
in maintaining the karyotype and pluripotent characteristics of PSCs (Barbaric & Dear,
2009). The use of these factors helps to induce pluripotency; it does not truly mimic
in vivo growth conditions.
Conventional cell culture of PSCs uses flasks or plates in a static environment.
Static culture has inherent drawbacks – cell growth is limited to the surface area of the
vessel and prevents the cells from rapidly growing. As each vessel has a slightly differ-
ent microenvironment, it is possible for the generation of heterogeneous subpopulations
of cells to occur during iPSC generation. The development of bioreactors as a culturing
mechanism has occurred to alleviate some of the problems associated with static cell
culture. A bioreactor generates a dynamic environment in which cells are capable of
growth. Research efforts have recently been focused on the study of stirred suspension
bioreactors for the growth and expansion of PSCs.

Stirred suspension bioreactors

Bioreactors have been used in a wide variety of biotechnological applications such as
the production of virus and protein, as well as in cell culture for the expansion of
embryonic stem cells (Martin, Wendt, & Heberer, 2004; Cormier, zur Nieden, Rancourt,
& Kallos, 2006). Bioreactor systems offer advantages over static culture flasks for ESC
26 B. Day and D.E. Rancourt

expansion, such as a homogenous culture environment (Chu and Robinson, 2001).

Bioreactors have the ability to monitor and fully control culture conditions such as
oxygen concentration, pH and temperature (Chu and Robinson, 2001). These systems
also allow for rapid expansion and production of large numbers of cells with minimal
labour input.
A stirred suspension bioreactor (SSB) uses an impeller physically to stir culture
media. In this system, PSCs form small aggregates of undifferentiated cells
(Cormier et al., 2006; Shafa et al., 2012). The level of hydrodynamic shear stress
generated by the rate in which the suspension is stirred is critical for PSC survival
(Cormier et al., 2006). For effective cell expansion, rotational speeds producing 6
dyne/cm2 of stress generate optimal proliferation and aggregate size. Rotation above
this level leads to smaller, damaged aggregates. Amalgamation of cells into larger
aggregates occurs when shear stress is below this level. Shear stress is used to con-
trol the size of aggregates allowing for maximum diffusion of nutrient mass transfer
in order to allow for all cells to receive adequate supply of oxygen and nutrients
(Kallos and Behie, 1999). Cells grown as aggregates in an SSB are capable of
expansion while maintaining pluripotency (zur Nieden, Cormier, Rancourt, & Kallos,
2007). Cells grown within the bioreactor do not differentiate even if treated to do
so with the removal of LIF (our unpublished data).
Shafa et al. (2012) have shown that SSBs can be used to derive and grow iPSCs
more efficiently than the traditional static culture methods. Here, terminally differenti-
ated MEFs were treated with a retrovirus containing Oct4, Sox2, Klf4 and c-Myc. The
cells were grown for 2 days in an adherent culture and transferred to SSBs for further
growth. The use of SSBs produced pluripotent iPSCs after only 12 days of growth
compared with the typical 30 days with static culture. This is one of the first studies to
demonstrate the potential of the bioreactor in a generation of iPSCs and subsequent
growth of PSCs. Additional studies have shown that as the cell is reprogrammed its
metabolism changes from reliance on oxidative cellular respiration pathway to a more
glycolytic one.

Metabolic profile of pluripotent cells

Glycolysis is a major metabolic pathway that converts glucose into pyruvate. The
energy released in this conversion is used to form adenosine-5'-triphosphate (ATP) and
nicotinamide adenine dinucleotide (NADH). The series of reactions in glycolysis pro-
duces intermediate monosaccharide compounds. These can act as an entry point for the
conversion of ulterior carbon sources such fructose and galactose for the cell to utilize.
The process is regulated by inhibiting or activating enzymes that are involved with gly-
colysis and typically based on changes in the free energy of each available step. Glycol-
ysis or a variant occurs within nearly all organisms in both aerobic and anaerobic
conditions. Under aerobic conditions cells will use the products of glycolysis, pyruvate
and NADH, to further produce ATP through cellular respiration.
Cellular respiration is a well-known metabolic pathway that converts larger
molecules into ATP through catabolic reactions. The process produces significantly
more free energy for a cell to use than glycolysis. As this takes place in the
mitochondria, it is characterized with a significantly increased mitochondrial activity.
This activity as well as enzyme expression and oxygen consumption can be used to
determine a cell’s metabolic profile. A metabolic profile can be used to understand
better how a cell functions in its niche and what processes are active.
 Biotechnology and Genetic Engineering Reviews 27

Embryonic stem cells

In the embryo prior to implantation and vascularisation, ESCs are exposed to hypoxic
environment conditions, with an oxygen partial pressure below 40 mmHg (Fischer and
Bavister, 1993). Cells grown in hypoxic conditions lack the ability to produce ATP via
cellular respiration; therefore they are reliant on glycolysis and anaerobic metabolism
for energy production. As the majority of cellular respiration takes place in the
mitochondria, ESCs have very few, or mitochondria with immature morphology. As
they differentiate, the number and morphology of the mitochondria change, allowing
for an increased production of ATP for use as terminally differentiated cells.
Cho et al. (2006) examined mitochondria of hESCs undergoing spontaneous differ-
entiation and observed a marked increase in the mass and DNA of mitochondria. This
also corresponded with expression of antioxidant enzymes and a four-fold increase in
the intracellular ATP levels within the cell. They concluded that differentiation of
hESCs resulted in dynamic changes in mitochondrial behaviour, demonstrating a shift
from an ESC-like metabolic profile to a drastically different progeny profile. Similarly
Kondoh et al. (2007) were able to show a direct correlation between a glycolytic profile
and proliferative capacity of mESCs. They found that proliferating ESCs typically had
higher activity of glycolytic enzymes, elevated glycolytic flux and a lower oxygen con-
sumption rate. This report revealed the relationship between ESCs and a low oxidative
profile.
Varum et al. (2009) were able to show that specifically targeting the mitochondria
using antimycin A to block complex III promoted pluripotency. This was demonstrated
through a two-fold induction of Nanog in antimycin A-treated cells with a correspond-
ing reduced expression of genes associated with differentiation. Interestingly, antimycin
A was able to replace bFGF in ESC culture media to maintain pluripotency. Their
results indicate that repressing mitochondrial function or manipulating glycolic over
cellular respiratory pathways may be a useful way to modulate ESC pluripotency. In a
different study, Varum et al. (2011) compared the metabolic profile of hESCs, iPSCs
and somatic cells and found that, while not identical, iPSCs behave very similar to
ESCs. The study tracked mitochondria localization and morphology, while analyzing
gene expression of several metabolic pathways, oxygen consumption rates and
intracellular ATP levels. This revealed that pluripotent cells rely mainly on glycolysis to
produce ATP and demonstrates the similarity between ESC and iPSC metabolic
profiles.

Induced pluripotent stem cells

Recently, iPSCs have emerged as a new field in stem cell research. These cells
have particular interest in regenerative medicine for the potential to produce patient-
specific pluripotent stem cells which would then be differentiated into a myriad of
tissues. The hope is that by using iPSCs there will be a reduction in immunologi-
cal rejection by the patient. These cells also avoid ethical issues raised by the
destruction of embryos as with ESCs. As a molecular tool, iPSCs also allow for
study of differentiation and pluripotency by examining gene expression and
metabolic profiles of terminally differentiated cells, reprogramming them and then
comparing the differences.
Similar to Varum and group, Prigione, Fauler, Lurz, Lehrach, & Adjaye (2010) were
able to show that while not identical, there is a distinct similarity between iPSC
and ESC metabolic profiles. The study demonstrates that mitochondria of somatic cells
28 B. Day and D.E. Rancourt

convert to a more pluripotent-like morphology and distribution through cellular

reprogramming. This results in a corresponding decrease in mitochondrial biogenesis,
DNA and intracellular ATP levels. They also looked at the global transcriptome profile
and were able to show a similar metabolic expression pattern of gene regulation in
mitochondrial biogenesis and metabolic genes. Armstrong et al. (2010) were able to
demonstrate that reprogramming reset oxidative stress levels by measuring reactive
oxygen species levels in iPSCs to a level similar to ESCs. This also suggests a
similarity between iPSC and ESC metabolic profiles and how the cells cope with
oxidative stress.
The culture conditions of PSCs have been explored further, mainly to improve
iPSC reprogramming efficiency. The culture conditions that best grow iPSCs also
support a more glycolytic metabolic profile. These are typically hypoxic conditions.
Yoshida, Takahashi, Okita, Ichisaka, and Yamanaka (2009) reported that 5% oxygen
conditions would significantly increase the efficiency of induction and generation of
iPSCs when grown in static culture. Growing in a hypoxic condition was not found to
change the differentiation potentials of iPSCs. This suggests a growing condition that
supports glycolysis and represses mitochondrial respiration would be ideal for PSC
growth. These are similar conditions to those used by Ezashi, Dash, and Roberts
(2005), who were able to repress spontaneous differentiation of ESCs using hypoxic
culture conditions.

Oxidative status and bioreactor growth

Previous studies have shown that PSCs grown within an SSB expand rapidly while still
maintaining their pluripotency. This is also true for the reprogramming of somatic cells
into iPSCs. While the mechanism for induced pluripotency in the SSB is unknown at
present, it is likely that the dynamic environment plays a large role. Based on the
design of the bioreactor, cells are grown in a hypoxic condition. As the conditions of
the bioreactor are fairly well defined, it is possible to maintain hypoxic growth condi-
tions. Cells that prefer glycolysis to grow are able to maintain pluripotency using this
pathway and expand rapidly owing to the abundance of nutrients found within an SSB.
As they move around in aggregates, cells are unable to overgrow in a particular area
and are still able to maintain adherence-based growth through cell–cell interactions.
Although cell expansion in the SSB is now studied in lab scale (i.e., 100 ml), the use
of glycolytic metabolism holds great promise for the further scaling up of cultures to
larger volumes.
Scale-up in bioreactor is based on the need for producing large enough quantities of
PSC in regenerative medicine. These methods are dependent on a readily available
source of high quality PSCs. It will be imperative to be able to control culture
conditions while producing vast amounts of cells. At present, there are a number of
physical concerns and limitations on the SSB when the volume is increased from the
current 100 ml to 1-liter volumes. At higher volumes vessel and impeller geometry is
not simply multiplied by a scaling factor of 10. These parameters affect the mass trans-
fer of oxygen and change mixing and shear stress in non-linear ways. It is expected
that, as the volume of the bioreactor increases, the environment will become more hyp-
oxic as surface aeration may fail to maintain a suitable oxygen level. It will be impera-
tive that PSCs are capable of surviving natural hypoxic conditions as levels of oxygen
are more difficult to maintain as the bioreactor expands.
 Biotechnology and Genetic Engineering Reviews 29

Conclusions
Recent studies have shown the capability of PSCs not only to grow in stirred suspen-
sion bioreactors but also to thrive. Furthermore, the study of the metabolic profile of
PSCs has revealed that cells use glycolytic pathways for ATP production. This is dem-
onstrated by the lack and underdevelopment of mitochondria in pluripotent cells, lower
levels of intracellular ATP and through the gene profile of pluripotent and differentiate
cells. The bioreactor artificially creates a hypoxic environment for the cells to grow
through surface diffusion of oxygen. Cells are capable of exploiting these conditions
along with an abundance of nutrients to grow rapidly while maintaining pluripotency.

References
Armstrong, L., Tilgner, K., Saretzki, G., Atkinson, S. P., Stojkovic, M., Moreno, R., … Lako, M.
(2010). Human induced pluripotent stem cell lines show stress defense mechanisms and
mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells, 28,
661–673.
Barbaric, I., & Dear, T. N. (2009). Culture of murine embryonic stem cells. Methods in molecular
biology, 561, 161–184.
Cho, Y. M., Kwon, S., Pak, Y. K., Seol, H. W., Choi, Y. M., Park do, J., … Lee, H. K. (2006).
Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the
spontaneous differentiation of human embryonic stem cells. Biochemical and Biophysical
Research Communications, 6, 1472–1478.
Chu, L., & Robinson, D. K. (2001). Industrial choices for protein production by large-scale cell
culture. Current Opinions in Biotechnology, 12, 180–187.
Cormier, J. T., zur Nieden, N. I., Rancourt, D. E., & Kallos, M. S. (2006). Expansion of
undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors.
Tissue Engineering, 12, 3233–3245.
Evans, M. J., & Kaufman, M. H. (1981). Establishment in culture of pluripotent cells from mouse
embryos. Nature, 292, 154–156.
Ezashi, T., Das, P., & Roberts, R. M. (2005). Low O2 tensions and the prevention of differentia-
tion of hES Cells. Proceedings of the National Academy of Science of the United States of
America, 102, 4783–4788.
Fischer, B., & Bavister, B. D. (1993). Oxygen tension in the oviduct and uterus of rhesus
monkeys, hamsters and rabbits. Journal of Reproduction and Fertility, 99, 673–679.
Kallos, M. S., & Behie, L. A. (1999). Inoculation and growth conditions for high-cell-density
expansion of mammalian neural stem cells in suspension bioreactors. Biotechnology and
Bioengineering, 63, 473–483.
Kondoh, H., Lleonart, M. E., Nakashima, Y., Yokode, M., Tanaka, M., Bernard, D., … Beach, D.
(2007). A high glycolytic flux supports the proliferative potential of murine embryonic stem
cells. Antioxidants & Redox Signaling, 9, 293–299.
Kurosawa, H., Kimura, M., Noda, T., & Amano, Y. (2006). Effect of oxygen on in vitro
differentiation of mouse embryonic stem cells. Journal of Bioscience and Bioengineering,
101, 26–30.
Martin, I., Wendt, D., & Heberer, M. (2004). The role of bioreactors in tissue engineering. Trends
in Biotechnology, 22, 80–86.
Naveiras, O., & Daley, G. Q. (2006). Stem cells and their niche: A matter of fate. Cellular and
Molecular Life Science, 63, 760–766.
Niwa, H., Ogawa, K., Shimosato, D., & Adachi, K. (2009). A parallel circuit of LIF signalling
pathways maintains pluripotency of mouse ES cells. Nature Letters, 460, 118–1122.
Prigione, A., Fauler, B., Lurz, R., Lehrach, H., & Adjaye, J. (2010). The senescence-related
mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells.
Stem cells, 28, 721–733.
Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A., & Benvenisty, N. (2000). Effects
of eight growth factors on the differentiation of cells derived from human embryonic stem
cells. Proceedings of the National Academy of Science of the United States of America, 97,
11307–11312.
30 B. Day and D.E. Rancourt

Shafa, M., Day, B., Yamashita, A., Meng, G., Liu, S., Krawetz, R., & Rancourt, D. E. (2012).
Derivation of iPSCS in stirred suspension bioreactors. Nature Methods, 8, 465–466.
Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell, 126, 663–676.
Varum, S., Momčilović, O., Castro, C., Ben-Yehudah, A., Ramalho-Santos, J., & Navara, C. S.
(2009). Enhancement of human embryonic stem cell pluripotency through inhibition of the
mitochondrial respiratory chain. Stem Cell Research, 3, 142–156.
Varum, S., Rodrigues, A. S., Moura, M. B., Momcilovic, O., Easley, C. A. 4th, Ramalho-Santos,
J., … Schatten, G. (2011). Energy metabolism in human pluripotent stem cells and their
differentiated counterparts. PLoS One, 6, e20914.
Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., … Gough,
N. M. (1988). Myeloid leukaemia inhibiting factor maintains the developmental potential of
embryonic stem cells. Nature, 336, 684–687.
Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., & Yamanaka, S. (2009). Hypoxia enhances
the generation of induced pluripotent stem cells. Cell Stem Cell, 5, 237–241.
zur Nieden, N. I., Cormier, J. T., Rancourt, D. E., & Kallos, M. S. (2007). Embryonic stem cells
remain highly pluripotent following long term expansion as aggregates in suspension
bioreactors. Journal of Biotechnology, 129, 421–432.