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Cellular Bioenergetics in Health and Diseases: New Perspectives in Mitochondrial Biology, 2012: 195-215
ISBN: 978-81-308-0487-3 Editors: Phing-How Lou and Natalia Petersen
6. Mitochondrial involvement in stemness
and stem cell differentiation
Anaïs Wanet, Thierry Arnould and Patricia Renard
Laboratory of Biochemistry and Cell Biology (URBC), NARILIS (Namur Research Institute for
Life Sciences), University of Namur (FUNDP), Belgium
Abstract. Mitochondria are known to exhibit different abundances,
morphologies and functions in different cell types that adapt their
number and activity in response to environmental and cellular cues.
Interestingly, a number of recent studies have highlighted changes in
mitochondrial content, architecture and function during the
differentiation of stem cells, as well as during the reprogramming of
somatic stem cells into induced pluripotent stem cells. Indeed, while
pluripotent cells generally contain fewer mitochondria and rely mainly
on glycolysis to meet their energy demand, differentiated cells display
a more developed mitochondrial network and rely on oxidative
phosphorylation for most of their energy production. Moreover, these
mitochondrial changes would also contribute to the differentiation
process itself. These observations strongly suggest the existence of an
interplay between mitochondrial biogenesis and stem cell
differentiation. In this chapter, we review the current knowledge about
the involvement of mitochondria in the stemness and differentiation
abilities of different stem cell types such as embryonic stem cells,
induced pluripotent stem cells and somatic stem cells.
Introduction
During the last few years, a growing interest has been devoted to the
study of the mitochondrial morphology, dynamics and functions in stem cells.
Correspondence/Reprint request: Dr. Anaïs Wanet, Laboratory of Biochemistry and Cell Biology (URBC)
NARILIS (Namur Research Institute for Life Sciences), University of Namur (FUNDP), Belgium
E-mail: [email protected]
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Indeed, accumulating data have highlighted the different mitochondrial
phenotypes in pluripotent and differentiated cells and suggested that
mitochondrial biogenesis would contribute to and/or be regulated by cell
differentiation. Indeed, while pluripotent cells display an « immature »
mitochondrial network (characterized by mitochondria with poorly developed
cristae and translucent matrices) and preferentially use anaerobic metabolism
for their energy supply, differentiated cells tend to exhibit a more developed
mitochondrial network and to rely mainly on oxidative phosphorylations to
produce ATP [1].
Stem cells are defined by two key properties: self-renewal, i.e. the ability
to proliferate without lineage commitment, and the capacity to differentiate
into one or more specialized cell types [2, 3]. Although many stem cell types
can be distinguished based on their pluripotency degree, stem cells can be
gathered in three categories: embryonic stem cells (ESCs), somatic (or adult)
stem cells (SSC) and induced pluripotent stem cells (iPSCs).
ESCs, which arise from the inner cell mass of the early blastocyst, have the
potential to generate any cell type derived from all three germ layers (endoderm,
mesoderm and ectoderm) and therefore exhibit the highest degree of pluripotency
[2, 3]. SSC, on the other hand, display reduced self-renewal and pluripotency
compared to ESCs [2]. Among this category are included – among others –
hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). While
ESCs and SSCs are natural stem cells, iPSCs are mature adult cells (such as
fibroblasts) that have been artificially reprogrammed to an ESC-like state by
overexpressing master « stemness » regulators such as Oct4, Nanog, Sox2,
KLF-4 and/or c-Myc [2, 4, 5]. Studies performed on these three stem cell
categories brought evidence of changes in mitochondrial morphology, dynamics
and/or functions during stem cell differentiation and, interestingly, suggested an
interplay between « stemness », stem cell differentiation and mitochondrial
biogenesis.
1. The interplay between mitochondrial biogenesis and cell
differentiation: The ESCs perspective
1.1. A mitochondrial «maturation» is observed during ESC
differentiation
During the last decade, several analyses of the mitochondrial morphology
using electron microscopy revealed a particular mitochondrial phenotype in
mouse and human ESCs, characterized by few mitochondria with poorly
developed cristae [1, 6-8]. Intriguingly, St. John and colleagues reported that
the mitochondria of a human ES cell line tend to localize perinuclearly [9], an
observation which was verified in mouse and other human ESC lines [10-13]
Mitochondrial involvement in stemness and stem cell differentiation
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but whose physiological significance is still obscure. Several studies
demonstrated that both the mitochondrial mass and mitochondrial DNA
(mtDNA) content increase during hESC differentiation, induced by the
retrieval of the feeder layer, FGF-2 (or b-FGF) and serum replacement, or
induced by embryoid bodies formation (depending on studies) [12, 14, 15].
These observations should nevertheless be moderated by the size of the cell
types compared, as recent data indicate that when considered relative to the
total cell protein content, ESCs (and iPSCs) and differentiated cells would
actually display similar mitochondrial mass ratio [16]. More interestingly
nevertheless, the mitochondria of differentiated cells display a larger
morphology and more distinct cristae [9, 13, 14] and these changes in
mitochondrial morphology are accompanied by an increase in ATP content
and reactive oxygen species (ROS) levels [14] (Figure 1). Besides, in a
specific model of cardiac differentiation, it was shown that mESC
differentiation is accompanied by the restructuration of the glycolytic
transcriptome and a shift from glycolysis to mitochondrial respiration [17],
suggesting that the changes in mitochondrial morphology are accompanied
by metabolic modifications during ESC differentiation. Supporting this
notion, ESCs were shown to depend mainly on glycolysis for ATP
production, and to produce minimal ATP amounts by OXPHOS in
comparison with differentiated cells (fibroblasts) [16]. Moreover, ESCs
induced to differentiate by retinoic-acid exposure display a gradual decrease
in glycolysis, reinforcing the concept of a metabolic reprogramming during
differentiation.
Regarding the mitochondrial membrane potential of ESCs and
differentiated cells, divergent data were obtained. On one hand, St John and
colleagues observed a lower proportion of mitochondria with a high
mitochondrial membrane potential in hESCs compared with differentiated
cardiomyocytes, which could indicate a lower oxidative metabolism in ESCs
[9]. On the other hand, Prigione et al. revealed a more elevated mitochondrial
membrane potential in hESCs (and in iPSCs) than in differentiated cells,
which is interpreted as the result of a reduced ATP consumption and a
glycolysis-based energy metabolism [18]. The use of different ES cell lines,
of various differentiation protocols, different culture conditions as well as
the pluripotency state and differentiation potential of the ESCs studied might
be partly involved in the discrepancies between these observations. Indeed,
by analyzing mESCs sorted for low or high resting mitochondrial membrane
potential, Schieke and colleagues demonstrated that mESCs with low
mitochondrial membrane potential differentiate efficiently in cells of the
mesodermal lineage but fail to efficiently form teratomas (which is an in vivo
measure of their pluripotency), whereas mESCs with high mitochondrial
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Figure 1. Embryonic stem cells and differentiated cells display different
mitochondrial morphology and functions. While the mitochondria of ESC are
characterized by a punctate, perinuclear arrangement, an electron-lucid matrix and
poorly developed cristae, mitochondria of differentiated cells form more developed
networks, have an electron-dense matrix and developed cristae. These morphological
modifications are accompanied by a shift from a glycolytic-based metabolism in ESCs
towards increased oxidative phosphorylation in differentiated cells. Conversely, an
opposite change for most of these features is observed during the reprogramming of
somatic cells.
membrane potential exhibit the opposite behavior [19]. Further studies are
nevertheless required to determine (1) if variations in the mitochondrial
membrane potential are observed during cell differentiation, and if these
variations are linked to the commitment to a specific lineage or are a
hallmark of cell differentiation; (2) what are the physiological reasons
underlying the change in mitochondrial membane potential and (3), what are
the molecular actors involved in mitochondrial membrane potential
regulation and variations. In relation with this last question, it was
demonstrated in a recent study that, while the mitochondrial membrane
potential of differentiated cells is maintained by the electron transport chain,
that of ESCs depends on glycolysis and the ATP hydrolase activity of the
F1F0-ATPase. Interestingly, the maintenance of the mitochondrial membrane
Mitochondrial involvement in stemness and stem cell differentiation
199
potential through glycolytic ATP hydrolysis in pluripotent cells would be
required for their viability and proliferation capacity [16].
Surprisingly, although both the mitochondrial mass and mtDNA copy
number are increased during hESC differentiation, the protein levels of
TFAM, PGC-1α and NRF-1, which are all three key regulators of
mitochondrial biogenesis, are decreased [14]. In line with this observation,
the mRNA levels of TFAM, PGC-1α and PGC-1β, Polγ and Polγ2, TFBM1
and TFBM2 and NRF1 (depending on studies) are reported to decrease
during the spontaneous in vitro differentiation of hESCs into embryoid
bodies, as well as the nuclear genes coding for the mitochondrial electron
transport chain (ETC) complex subunits [12, 20, 21]. However, the transcript
expression levels of ETC-coding genes and of several mitochondrial
biogenesis regulators (TFAM, Polγ, PGC-1α and PGC-1β) are increased in
fully differentiated teratomas, representing an in vivo model of
differentiation. Although these latter results may be biased by the cancerous
features of teratomas, these data underline the importance of considering the
level of differentiation, which is less mature in embryoid bodies than in
teratomas [21]. One can also expect that tissue- or cell type-directed
differentiations of ESCs or iPSCs might provide a still different picture of the
regulatory networks of mitochondrial biogenesis. Although this hypothesis
needs to be investigated, the decreased expression of the mitochondrial
biogenesis regulators observed in in vitro differentiating ESCs has been
proposed to represent a nuclear response to the decreased amount of mtDNA
in pluripotent cells [21] (one can refer to this book section devoted to the
«mitochondrial retrograde communication»). If true, this hypothesis raises a
still unresolved question: how is mitochondrial biogenesis prevented in
pluripotent cells expressing high levels of these regulators?
In order to correlate the kinetics of mitochondrial biogenesis and network
formation with the loss of pluripotency and the differentiation process,
Mandal and colleagues analyzed the changes in mitochondrial morphology
and the expression of OCT4 and NANOG during the differentiation of HSF1
cells (a hESC line), induced by the retrieval of the feeder layer, of FGF-2 and
serum replacement. They found that the formation of the mitochondrial
network is initiated while the cells are still expressing the pluripotency
markers, and that an extensive mitochondrial network has developed by the
time OCT4 and NANOG expressions are lost. Accordingly, an increase in
ATP and ROS levels is seen during the course of differentiation [10].
However, a study performed on differentiating mouse ESCs (mESCs) (using
a differentiation protocol based on embryoid bodies formation) demonstrated
that the expression of several pluripotent markers (Dppa5, developmental
pluripotency associated gene; Pramel7, Prame-like 7 and Ndp5211 or
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Calcoco2) decreases before the increase in mtDNA copy number, suggesting
that mtDNA would be extensively replicated after the commitment of mESCs
to a specific cell fate [15]. Although the different data obtained may result
from the different experimental settings (such as the pluripotency markers
analyzed, the way differentiation is triggered and the methods used to
evaluate mitochondrial biogenesis) and cell types, further studies seem
necessary to determine what is the event, between the loss of pluripotency or
mitochondrial biogenesis, that occurs first, and if this chronology is
conserved among species, stem cell types and differentiation protocols.
1.2. Mitochondrial biogenesis not only correlates with ESC
differentiation, but could also participate in it
The use of different chemical molecules has provided further evidence
about the involvement of mitochondrial biogenesis and function in stem cell
differentiation. When mitochondrial function is uncoupled in mESCs and
hESCs by carbonyl cyanide m-chlorophenylhydrazone (CCCP, a
protonophore depolarizing the inner mitochondrial membrane, resulting in
uncoupled oxidative phosphorylation) in mESCs and hESCs, the
transcriptional programs necessary for the embryonic lineage differentiation,
and especially HOX genes expression, are repressed, suggesting that
attenuated mitochondrial function results in compromised differentiation. On
the other hand, CCCP-treated mESCs and hESCs demonstrate enhanced
glycolytic metabolism and increased expression of OCT4, NANOG and
SOX2 compared with the untreated controls, suggesting that the pluripotency
of mESCs and hESCs is maintained, or even enhanced upon CCCP
uncoupling. It needs to be mentioned, nevertheless, that the proliferation of
self-renewing mESCs and hESCs is slowed down upon CCCP treatment,
which suggests that these cells may rely, at least partly, on mitochondrial
functions for their proliferation [10].
Regarding hepatogenic differentiation, a robust increase in PPAR-β
expression (the involvement of PPAR members in mitochondrial biogenesis,
as well as that of other mitochondrial biogenesis inducers, is developed in a
chapter of this book devoted to mitochondrial biogenesis) is observed during
the late differentiation course of mESCs. Interestingly, a PPAR-β agonist
significantly increases mitochondrial abundance and the number of albuminpositive cells differentiated from mESCs. On the opposite, both hepatogenic
differentiation and mitochondrial biogenesis are hampered in the presence of
a PPAR-β antagonist. PPAR-α and PGC-1α, on the other hand, display both
transient increased expressions during the early phase of hepatocyte
differentiation. However, although stimulating mitochondrial biogenesis, a
Mitochondrial involvement in stemness and stem cell differentiation
201
PPAR-α agonist does not increase hepatocyte differentiation. Together, these
results suggest that PPAR-α-induced mitochondrial biogenesis would only be
necessary for the early phase of mitochondrial biogenesis, while PPAR-β
would play a more important role in the control of cellular energy
metabolism during hepatocyte differentiation and maturation [22]. S-NitrosoN-AcetylPenicillamine (SNAP), a NO donor, also stimulates the mitochondrial
biogenesis and enhances the hepatogenic differentiation of mESCs [23], as
assessed by the measurement of urea and albumin secretion, cyp7a1 promoter
activity and glucose and lactate metabolisms, which are increased during
hepatocyte differentiation and maturation. These results further support the
involvement of mitochondrial biogenesis in the hepatogenic differentiation of
mESCs.
A specific role for the complex III of the mitochondrial electron transport
chain in ESC differentiation was suggested by several groups using
antimycin A, a molecule blocking the electron flow in the complex III.
Indeed, the treatment of mESCs with this inhibitor results in a blockade of
heart cell differentiation, while the use of complex II and complex IV
inhibitors (thenoyltrifluoroacetone and potassium cyanide) does not prevent
cardiomyocyte differentiation. This suggests that the activity of complex III
is necessary, while those of complex II and IV are dispensable, for heart cell
differentiation. As antimycin A treatment inhibits spontaneous intracellular
Ca++ oscillations and as a pulse of ionomycin (an ionophore used to raise the
intracellular level of Ca++), given at an appropriate time, restores
cardiomyocyte differentiation, mitochondrial complex III activity has been
suggested to be required for the differentiation of cardiomyocytes through its
involvement in Ca++ oscillations [24]. More recently, it appeared that
complex III activity is not only required for cardiomyocyte differentiation,
but also involved in stem cell pluripotency. Varum et al. showed that the
treatment of hESCs with antimycin A results in an increased expression of
NANOG (OCT4 mRNA levels remain unchanged), maintains the ESC ability
to form teratomas exhibiting tissues of all three germ layers (which suggests
that treated ESCs are still pluripotent), and results in the repression of genes
associated with differentiation. Interestingly, they demonstrated that the ROS
generation occuring at complex III upon antimycin A treatment is at least
partially responsible for the upregulation of NANOG expression in these
cells, implying a role of ROS in governing stemness and differentiation [25].
The involvement of ROS in stem cell differentiation is further supported
in the model of cardiomyocyte differentiation of ESCs, although in an
opposite fashion. Indeed, it was shown that ESCs cultured in physiological
glucose concentration (5 mM) display an altered metabolism, decreased ROS
levels and fail to generate cardiomyocytes whereas ESCs maintained in
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supraphysiological glucose concentration (25 mM) exhibit an opposite
behavior. Besides, the outcome of ESCs cultured in low-glucose medium is
reversed when the medium is supplemented with ascorbic acid, paradoxically
acting as a pro-oxidant, or when an upstream p38 MAPK kinase (MKK6) is
expressed, and those of ESCs cultured in high-glucose medium are reversed
when cells are exposed to antioxidant treatment. These obervations suggest
that supraphysiological levels of glucose are required for cardiomyocyte
formation through ROS-dependent p38 activation [26]. Therefore, we may
speculate that ROS regulate both stemness and differentiation, depending on
their levels, the cell types and differentiation models studied.
1.3. Several mitochondrial and mitochondria-related proteins
have been involved in the mitochondrial modifications occuring
during ESC differentiation
Beside the use of chemical compounds to increase or inhibit
mitochondrial content or function, several authors analyzed the modifications
in the expression of mitochondria-related proteins on ESC pluripotency and
differentiation. These studies have pinpointed a possible involvement of Polγ,
Gfer (growth factor erv1-like), Ptpmt1 (Pten-like phosphatidylinositol
phosphate (PIP) phosphatase, mitochondrial 1) and UCP2 (uncoupling
protein 2) in governing stemness and differentiation.
Indeed, steady-state levels of Polγ, the DNA polymerase responsible for
mtDNA replication (see this book chapter devoted to mitochondria
biogenesis), are suggested to be necessary for the maintain of mESC
pluripotency, as both up- and downregulation of Polγ mRNA are observed
during the onset of differentiation of different mESC types, and as Polγ
knockdown in mESCs results in reduced OCT4 protein levels and increased
brachyury levels (a specific marker of the mesodermal lineage) [15].
The growth factor Gfer, a FAD-dependent sulfhydryl oxidase
predominantly localized in the intermembrane space of the mitochondria, was
demonstrated to maintain mitochondrial dynamics and pluripotency of
mESCs through the modulation of Drp1 (dynamin-related protein 1)
expression [27]. Drp1 is a dynamin-like GTPase capable of self-assembly
into multimeric ring-like structures necessary to mitochondria fission [28].
The knockdown of Gfer in mESCs results in the reduced expression of
NANOG, OCT4 and SSEA1 (stage specific embryonic antigen 1), three
markers of mESC pluripotency, in diminished survival, in impaired embryoid
body formation and in the loss of mitochondrial function through an increase
in Drp1 levels, which results in enhanced mitochondrial fission and the loss
of mitochondrial membrane potential. On the opposite, Gfer overexpression
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is associated with an increased expression of NANOG and OCT4 and a
decrease in Drp1 levels, which impedes mitochondrial fission. Interestingly,
the effects of Gfer expression modulation on pluripotency gene expression
depend on Drp1 expression, as the expression of a dominant negative mutant
of Drp1 in Gfer-knockdown mESCs restores OCT4 and SSEA1 expression to
a level similar to control cells. Gfer may therefore regulate ESCs
pluripotency or stemness by regulating Drp1 expression and therefore
controlling mitochondrial dynamics. It is also worth mentioning that Gfer
knockdown in differentiated cells such as mouse embryonic fibroblasts does
not affect cell survival and mitochondrial content, morphology and function,
suggesting that Gfer would be dispensable for the survival and mitochondrial
functions of differentiated cells [27].
Ptpmt1, which is specifically localized in the mitochondrial inner
membrane, was also identified as specifically important for the maintain of
ESC ability to differentiate. The disruption of the gene encoding Ptpmt1 in
mice results in postimplantation lethality, impairs the proliferation of the
inner cell mass cells of blastocyts, decreases the proliferation and
differentiation of mESCs. Ptpmt1-depleted cells also display a decreased
oxygen consumption associated with enhanced glycolysis. Besides, the
depletion of Ptpmt1 in mESCs reduces fusion events, resulting in the
fragmentation of the mitochondrial network, an effect that would be linked to
the accumulation of PIP substrates such as PI(3,5)P2, as the perfusion of
PI(3,5)P2 in WT mESCs induces mitochondrial fragmentation in a similar
fashion than Ptpmt1 deficiency. Similarly than for Gfer, Ptpmt1 knockdown
in differentiated cells does not disturb cell function, suggesting that Ptpmt1 is
specifically important for stem cells [29].
In a recent report, UCP2 was identified as a central regulator of ESC and
iPSC energy metabolism. As previously mentioned, a metabolic switch from
glycolysis to oxidative phosphorylation was suggested to occur upon cell
differentiation. Interestingly, UCP2, which is overexpressed in pluripotent
cells when compared with differentiated cells or embryoid bodies, prevents
mitochondrial glucose oxidation and favors glycolysis through a substrate
shunting mechanism in pluripotent cells (UCP2 is proposed to block glucosederived pyruvate oxidation in mitochondria [30]). During cell differentiation,
UCP2 expression is repressed (as demonstrated in the model of retinoic-acidinduced differentiation of ESCs), which enables the transition from
glycolysis to glucose oxidation in mitochondria. Interestingly, the ectopic
expression of UCP2 in pluripotent cells induced to differentiate by retinoicacid exposure, impairs the induction of developmental gene expression,
suggesting that UCP2-mediated regulation of energy metabolism is required
for the early differentiation of pluripotent cells. Although UCP2 repression
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was demonstrated to contribute to the ROS accumulation seen in
differentiating cells, UCP2 would regulate cell differentiation by a still
unknown mechanism other than ROS (at least in the model studied), as the
use of ROS scavengers does not reverse the repression of differentiation seen
in pluripotent cells ectopically expressing UCP2 [16].
2. A « rejuvenated » mitochondrial phenotype is observed in
iPSCs, and is reversed during iPSC differentiation
Because of their potential therapeutic applications in regenerative
medicine, their advantages over ESCs in terms of immune rejection but also
in terms of ethical issues, a lot of attention has been paid to iPSCs in the aim
to evaluate to what extent these cells resemble ESCs. Several groups
therefore analyzed the mitochondrial morphology and functions during iPSC
reprogramming and differentiation.
In different studies, hESCs and hiPSCs (human iPSCs) were shown to
have comparable mitochondrial masses and genome copy numbers, both
lower than those of fibroblast-like cells differentiated from these hESCs and
hiPSCs and of control fibroblasts [12, 20]. Besides, even though a report
mentioned that hiPSC mitochondria display an intermediate phenotype
between hESCs and differentiated cells [13], another one found that hiPSCs
exhibit fewer mitochondria, localized at the two sides of the nuclei, and with
poorly developed cristae [12], which are characteristic features of ESC
mitochondria as described in the previous section. During the differentiation
of hESCs and hiPSCs, however, an increase in mtDNA copy number and in
the mitochondrial content is observed, and mitochondria (re)acquire an
elongated shape with developed cristae [12]. Interestingly, a recent report
mentioned that the reprogramming of aged fibroblasts (derived from a 84year-old woman) into iPSCs is associated with similar changes in
mitochondrial morphology compared to the reprogramming of young cells,
despite the presence of karyotype aberrations in the aged reprogrammed cells.
These observations suggest that the presence of chromosomal alterations in
aged somatic cells does not prevent their reprogramming into iPSCs neither
the associated changes in mitochondrial morphology [18].
Further supporting their similarities, both hESCs and hiPSCs are
characterized by similar low ROS levels compared with adult human dermal
fibroblasts, and both cell types produce higher ROS levels during differentiation
[12, 20]. In addition, both hESCs and hiPSCs display lower ATP levels and
higher lactate production compared with fibroblasts [12, 13], whereas ATP level
increases and lactate production decreases during hESC and hiPSC differentiation
Mitochondrial involvement in stemness and stem cell differentiation
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[12]. In agreement with these data, it was demonstrated that both hESCs and
hiPSCs rely less on OXPHOS than fibroblasts [13]. Instead, pluripotent cells
would rely on glycolysis to meet their energy demands. This hypothesis is
strengthen by the observation that pluripotent cells express higher protein levels
of hexokinase II compared with fibroblasts, and increased levels of
phosphorylated PDH E1α [13] that inactivates the pyruvate dehydrogenase
(PDH) complex and results in lower levels of substrates entering the TCA cycle
[31]. Intriguingly, hESCs and hiPSCs were found to express components of the
mitochondrial complexes II, III and V at higher levels than fibroblasts. Although
this hypothesis needs to be further explored, Varum et al. suggested that the
higher expression of c-Myc in hESCs and hiPSCs compared with fibroblasts may
be responsible for this effect [13]. Indeed, c-Myc, which is essential for the
maintenance of ESC self-renewal and used as a reprogramming factor during the
generation of iPSCs [32], is reported to promote mitochondrial biogenesis and the
expression of genes involved in mitochondrial structure and function [33].
As far as mitochondrial biogenesis regulators are concerned,
differentiating hESCs and hiPSCs also exhibit similar changes in their
expression. For instance, TFAM, Polγ, PGC-1, Polγ2, POLRmt, and TFBM1
mRNAs are all downregulated during the in vitro differentiation of both
hESCs and hiPSCs into embryoid bodies [12, 20, 21]. As mentioned earlier,
the higher expression of these mitochondrial biogenesis regulators in
pluripotent cells may represent a nuclear response to decreased amounts of
mtDNA [21]. Nevertheless, some differences were also noted for few genes,
including for TFBM2 and MTERF, which exhibit different trends during the
differentiation of hESCs and hiPSCs [20].
Altogether, these data strongly suggest that, although not identical, iPSCs
share similar mitochondrial morphology and function with ESCs and both
cell types exhibit similar changes in mitochondrial architecture and activity
during their differentiation. These findings suggest that when fibroblasts,
with a mature mitochondrial network, are reprogrammed into pluripotent
stem cells, the mitochondrial phenotype is also reversed towards an
« immature » phenotype, strongly suggesting that cell differentiation and
mitochondria maturation are intimately intricated. This raises an important
question : is it the cell differentiation process that governs mitochondria
maturation, and/or can we consider that mitochondria maturation allows the
cellular differentiation?
3. Mitochondrial biogenesis in somatic stem cell differentiation
Although fewer reports were published about the involvement of
mitochondria in SSC differentiation compared with ESCs and iPSCs, several
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studies nevertheless suggest a mitochondrial biogenesis and mitochondrial
contribution during SSC differentiation. The following sections will only
discuss the findings emerging from studies performed on MSCs and HSCs,
but increasing evidence also point toward a possible involvement of
mitochondrial biogenesis in the differentiation of other stem cell categories,
such as neural stem cells [34] and cardiac mesangioblasts [35].
3.1. Emerging data point toward a role of mitochondria in MSC
differentiation
Although no change was observed in the mitochondrial mass, studies
performed on hMSCs brought evidence of increased mtDNA copy number,
enhanced expression of the protein subunits of the respiratory enzymes,
increased oxygen consumption rate, and increased intracellular ATP content
during the osteogenic differentiation process [36]. As mentioned in the previous
section, Varum et al. reported a higher expression of the protein levels of
complexes II, III and V in hESCs and iPSCs than in fibroblasts [13] – that
might seem in contradiction with the expression changes observed by Chen et
al. Nevertheless, Varum and coworkers did not analyze the abundance of
these complexes upon hESC and iPSC differentiation – therefore, the
diverging data obtained may, at least partly, result from the use of different
cell lines to compare pluripotent and differentiated cells, and/or from the
different biology of ESCs, iPSCs and MSCs. Besides, although only studied
at the mRNA levels, the electron transport chain genes were shown to be
downregulated during the in vitro differentiation of ESCs and iPSCs, while
they tend to upregulate during in vivo ESC and iPSC differentiation (teratoma
formation) [21]. As mentioned earlier, although these observations may be
biased by the tumorigenic nature of teratomas, the level of differentiation
could also account for the discrepancies between these observations and
therefore, for the diverging results obtained by [36] and [13].
Human MSCs are also reported to be more dependent on glycolysis than
differentiated osteoblasts [36], a finding that is in agreement with the data
obtained in ESCs and iPSCs. However, unlike the expression patterns
observed in differentiating ESCs and iPSCs, the mRNA levels of TFAM,
PGC-1α and Polγ were found to increase during the differentiation of hMSCs
[36]. According to Prigione and Adjaye, these different observations may be
explained by different nuclear responses due to more or less abundant
mtDNA in different cell types [21], or they may also be linked to the features
of the differentiation model used. Indeed, the spontaneous in vitro
differentiation of ES cells into embryoid bodies [21] leads to the emergence
of cells committed into several cell types, but not fully differentiated, while
Mitochondrial involvement in stemness and stem cell differentiation
207
the population of cells undergoing a directed osteogenic differentiation
process [36] is less heterogenous and closer to the terminal differentiation
state.
Importantly, a study performed on ATSC cells, an adult rhesus macaque
stromal cell line isolated from adipose tissue and known to spontaneously
differentiate when cultured over a period of 20 passages, demonstrated that
the differentiation-related changes in mitochondrial properties may be used as
indicators of stem cell differentiation competence [11]. Indeed, a lower
proportion of cells of a later passage displays a perinuclear arrangement of
mitochondria, and these cells have higher ATP content and differentiate more
efficiently into adipocytes compared with cells from an earlier passage.
Therefore, the perinuclear distribution of mitochondria and a low ATP
content per cell are suggested to be indicators of stem cell differentiation
competence, while a shift from this profile to the one observed at later
passages may indicate that cells are differentiating or, possibly, becoming
senescent [11].
Supporting the notion that stem cell mitochondria may be involved in
their stemness status, it was found that the reprogramming of adult
cardiomyocytes toward a progenitor-like state, which occurs during their
partial fusion with hMSCs, depends on the transfer of stem cell mitochondria
into cardiomyocytes [37]. Further studies are nevertheless needed to elucidate
the role of stem cell mitochondria in cardiomyocyte reprogramming.
Regarding ROS, both increased and decreased ROS levels, depending on
the differentiation type studied, were found to be involved in MSC
differentiation. On one hand, a decrease in ROS levels was suggested to be
necessary for the osteogenic differentiation of human bone-marrow MSCs
(BM-MSCs). Indeed, the expression of catalase and manganese-dependent
superoxide dismutase (MnSOD, also known as SOD2) are increased in
osteoblasts compared to undifferentiated MSCs, resulting in a decrease in
intracellular ROS levels during the early phase of osteogenic differentiation.
Importantly, the exogenous administration of H2O2, as well as the treatment
of differentiating hMSCs with oligomycin, retard the osteogenic
differentiation process, further supporting the importance of reduced levels of
ROS (and, upstream of this, of induced catalase and MnSOD expression) for
the osteogenic differentiation [36]. Given that the MnSOD was identified as a
nucleoid complex component [38] and demonstrated to act as a mitochondrial
fidelity protein for the Polγ [39], one may wonder, while this has not been
studied so far, whether the MnSOD could not be also involved (beside its role
in decreasing ROS levels) in the mitochondrial biogenesis observed during
cell differentiation. On the other hand, Tormos et al. demonstrated that the
ROS produced by the mitochondrial complex III promote adipogenic
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differentiation of human BM-MSCs by inducing the expression of PPAR-γ
[40], the master regulator of adipogenesis [41]. Early in the adipogenic
differentiation process (two days after the initiation of the adipogenic
treatment), the oxygen consumption rate and ATP synthesis of BM-MSCs are
increased, as well as the intracellular concentration of H2O2. These ROS are
required for adipocyte differentiation, as adipocyte differentiation is
hampered in the presence of mitochondrial-targeted antioxidants, while the
addition of exogenous H2O2 rescues the differentiation process [40].
Similarly, the treatment of a MSC line with the antioxidant N-acetyl-lcysteine also blocks their differentiation into adipocytes [42]. It needs to be
mentioned, nevertheless, that the role played by ROS during the adipogenic
differentiation process is likely complex, as both differentiation-promoting
and differentiation-inhibiting effects were observed depending on studies [43,
44]. Whether these discrepancies are related to the use of different cell lines
and cell types, to a particular timing and/or to different differentiation
protocols remains to be determined. From a molecular point of view, during
the adipogenic differentiation of MSCs, the mitochondrial complex III was
demonstrated to be involved in the production of superoxide ions, which are
converted to H2O2, that initiate the PPAR-γ–dependent transcriptional
machinery driving adipocyte differentiation. Besides, it was shown that
mTORC1 (mammalian target of rapamycin complex 1) is involved in the
increased ROS levels during the initiation of adipocyte differentiation, and
that it would also drive adipocyte differentiation by acting on unidentified
effectors other than ROS. Therefore, the increase in mitochondrial
metabolism observed during differentiation would not only be necessary to
meet the energy demands of the differentiation process but would also be
required, through ROS production, for promoting the differentiation process
[40].
3.2. Mitochondrial features are also modified during HSC
differentiation
As seen for other pluripotent cell types, HSCs are characterized by a poor
mitochondrial content, spread in bipolar perinuclear clusters, a poor oxygen
consumption, a decreased expression of OXPHOS complex subunits per
mitochondondria and a weak OXPHOS activity [45]. Moreover, as observed
in ESCs, iPSCs and other SSCs, a mitochondrial biogenesis occurs during the
loss of pluripotency/differentiation of HSCs. CD34 is a specific surface
marker of hematopoeitic stem/progenitor cells whose expression is lost upon
HSC differentiation. Interestingly, Piccoli et al. observed an inverse
Mitochondrial involvement in stemness and stem cell differentiation
209
correlation between CD34 expression and mitochondrial content that might
indicate a shift toward a more efficient bioenergetic metabolism during the
onset of commitment [45]. However, in another study, devoted to long-term
repopulating HSCs, an increase in mitochondrial mass and mitochondrial
membrane potential was found in cells upregulating CD34 [46]. While in
apparent paradox with Piccoli and coworkers’ findings, these discrepancies
may be explained by the fact that CD34, while being a HSC marker, is poorly
expressed in long-term repopulating HSCs [46]. Therefore, a mitochondrial
biogenesis is observed when HSCs lose their long-term repopulating ability,
which is in agreement with the more general observation of a mitochondrial
biogenesis during cell commitment and loss of pluripotency.
In agreement with this correlation between mitochondrial biogenesis and
loss of pluripotency, it was observed that the deletion of the autophagy gene
Atg7 in the hematopoietic system results in an accumulation of mitochondria
with higher membrane potential, in an accumulation of ROS and DNA
damages as well as in the reduction of the number of progenitors of multiple
lineages [47]. These observations suggest, although not yet confirmed for
other types of stem cells, a role of mitophagy in the maintenance and
regulation of mitochondrial content and ROS production on the one hand,
and in the maintenance of HSC quality on the other hand.
The role of ROS in regulating HSC maintenance is supported by other
findings. Indeed, the conditional deletion of Tsc1 (tuberous sclerosis
complex), a negative regulator of mTORC1, results in mTOR
signaling pathway hyperactivation, drives a shift of HSC state from
quiescence to rapid cycling and results in decreased repopulation
capability of HSCs. Interestingly, Tsc1-deficient HSCs have increased
mitochondrial mass, mitochondrial DNA copy number and ROS levels. ROS
could thus be involved in regulating HSC quiescence, as the use of the
antioxidant N-acetylcysteine rescues the self-renewal ability of HSCs [48].
Beside mTORC1 signaling, another pathway, mediated by Lkb1 (liver
kinase B1 or serine-threonine kinase 11), has been involved in regulating
HSC quiescence and proliferation possibly through an effect on
mitochondrial functions. Indeed, Lkb1-deficient HSCs display decreased
repopulation abilities, are prone to exhaustion, downregulate PGC-1α and
have reduced mitochondrial DNA copy number, mitochondrial potential and
ATP levels [49]. These results, along with those of Chen et al. [48], suggest
the need of maintaining mitochondrial content and function in a certain range
to ensure HSC maintenance. Lkb1 loss has also been demonstrated to induce
apoptosis of HSCs and bone marrow cells, a process which is preceeded by
an autophagic survival response [50]. Interestingly, Lkb1 has been proposed
to be required for the maintenance of energy homeostasis in bone marrow
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cells, as its inactivation results, as previously mentioned, in a decrease in
mitochondrial membrane potential and ATP levels but also in a decrease in
basal mitochondrial oxygen consumption and total mitochondrial oxidative
capacity, despite an increased glucose uptake [50]. These effects of Lkb1 on
the cell cycle regulation, cell survival, mitochondrial function and energy
homeostasis, which are independent of mTOR signaling, depend both on
AMPK-dependent and on AMPK-independent mechanisms [51]. It needs to
be stressed however, as mentioned by Gurumurthy and colleagues, that it is
still unknown if these mitochondrial function defects are direct or secondary
consequences of Lkb1 inactivation, as the cell death program is rapidly
induced in Lkb1-deficient cells [50].
4. From development to disease: The mitochondria phenotype
of cancer stem cells
As described in the previous sections, changes in mitochondrial
abundance, morphology and functions are observed during the differentiation
of different stem cells types, ranging from embryonic stem cells to induced
pluripotent stem cell and somatic stem cells. Interestingly, the mitochondrial
phenotype observed in pluripotent cells would not only be a feature of normal
stem cells, but may also be used as an indicator of cancer stem cells (CSCs).
Indeed, lung cancer stem cells (LCSCs) isolated from the A549 lung cancer
cell line are characterized by mitochondria with a perinuclear arrangement, a
low mtDNA content, consume less oxygen and have reduced ATP and ROS
levels compared with non-LCSCs [52], suggesting that mitochondrial and
energy metabolism features could be used to determine the « stemness » of
both normal and cancer stem cells. Interestingly, an induction of mtDNA
abundance and mitochondrial mass is observed during the early
differentiation of LCSCs, reinforcing their similarity with normal stem cells.
Given the importance of CSCs in tumor progression and recurrence, it would
be of particular interest to study the mitochondrial properties of other CSC
types, to determine if the « immature » mitochondrial phenotype is a
hallmark of all CSCs.
5. Questions and future prospects
Considering the huge therapeutic potential offered by stem and
progenitor cells, as well as the necessity to get deeper insights into the
biology of CSCs in order to counteract cancer progression and recurrence, the
field of mitochondrial maturation during cell differentiation appears very
Mitochondrial involvement in stemness and stem cell differentiation
211
promising. Indeed, given the mitochondrial involvement in stemness and
differentiation described in the previous sections, one can ask whether
manipulating mitochondrial content and/or function, or mitochondrial-related
signaling pathways, couldn’t be used for the more efficient generation of
iPSCs or, on the opposite, for the more efficient differentiation of pluripotent
cells, which could be of particular interest for regenerative therapies. One
should bear in mind, however, several important facts and unresolved
questions before considering such a perspective.
First of all, although sharing the characteristic stemness properties, i.e.,
self-renewal and pluripotency, ESCs and iPSCs are not equivalent with
respect to proteomes and phosphoproteomes, transcriptomes and epigenetic
marks [53-56]. For instance, reprogrammed cells have been shown to retain
an « epigenetic memory » of their original tissue, and to exhibit a unique
gene expression signature independent of their origin or the method by which
they were generated [53-54]. Besides, it was shown that the reprogramming
of somatic cells into iPSCs is accompanied by alterations not only in the
nuclear genome [54], but also in the mitochondrial genome [57]. Therefore,
although these alterations do not prevent the reprogramming process, the
occurrence of pathogenic mutations (both in the nuclear and mitochondrial
genomes) in reprogrammed cells should be an important parameter to
monitor in the perspective of their use in cell-based therapy.
Secondly, the understanding of the role played by mitochondria during
stem cell differentiation is still in its early stages and requires more in depth
studies, focussing on an enlarged panel of stem cell types and differentiation
processes. It is generally accepted that mitochondria are phenotypically
different in stem cells than in differentiated cells, especially in terms of
metabolic activity, the stem cells relying essentially on a glycolytic
phenotype while differentiated cells are more oxidative. However, when
trying to depict more precisely the mitochondrial phenotype of stem cells
versus differentiated cells, and the regulatory pathways leading to
mitochondria “maturation” along differentiation, attention must be paid to
critical parameters: the type of stem cells, the spontaneous or directed
(specific) differentiation, as well as the level differentiation. To illustrate this,
it is instructive to examine two works: one dedicated to the osteogenic
differentiation of human bone marrow-derived MSCs [36], and the second
one to the spontaneous differentiation of ESCs and iPSCs [21]. For instance,
an unexpected downregulation of critical mitochondria biogenesis regulators
including PGC-1α, Polγ and TFAM has been observed upon in vitro
differentiation (embryoid bodies formation) of hESCs and hiPSCs, while
these regulators tend to be upregulated when the same cells are differentiated
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in vivo (teratoma formation) [21]. In line with this latter observation, the
same mitochondrial biogenesis regulators are clearly upregulated when
hMSCs undergo a directed differentiation towards osteoblasts [36]. On the
other hand, the different mitochondrial biogenesis markers do not necessarily
evolve in parallel. Although mtDNA content has been shown to increase with
cell differentiation in all the examined studies (and to the best of our
knowledge), this is not the case with some other mitochondrial markers like
nuclear-encoded subunits of the ETC. The expression of several proteins of
the ETC increase in hMSCs after osteogenic differentiation [36], while their
mRNA level is decreased in hESCs spontaneously differentiated into
embryoid bodies (the authors attributed this latter data to a high c-Myc
activity in ESCs and iPSCs, c-Myc being involved in the transcriptional
regulation of the genes coding for OXPHOS subunits) [21]. Whether this
contradiction is due to discrepancies between the transcript and protein
levels, which happens quite often, or to biological differences between the
two different models studied remains to be solved. However, if it is
confirmed that in ESCs the protein level of the nuclear-encoded ETC
subunits is downregulated upon differentiation, along with an increase in
mtDNA, then the observed metabolic oxidative switch might be due to the
critical role of the 13 mitochondria-encoded ETC subunits in the assembly of
respiratory supercomplexes (see this book chapter devoted to mitochondria
biogenesis).
Eventually, beside the specificities linked to cell types and differentiation
types and efficiencies, the general increase in mitochondrial biogenesis
and/or function occuring during the differentiation of different pluripotent
cell types leaves many open questions. For example, what are the
mechanisms restricting mitochondrial biogenesis and favoring a glycolytic
metabolism in stem cells? What are the signals controlling mitochondrial
biogenesis upon stem cell differentiation? How can an activated
mitochondrial biogenesis favor various differentiation processes, as
illustrated in the different previous sections? What are the molecular players
linking the mitochondrial biogenesis and the differentiation processes? Here
are so many questions whose answers should help to better understand the
biology of stem cells and, undoubtedly, the biology of the mitochondrion
itself.
Acknowledgements
A. Wanet is recipient of the doctoral fellowship from the Fonds National
pour la Recherche Scientifique (FNRS, Belgium). This work was supported Mitochondrial involvement in stemness and stem cell differentiation
213
by the Association Belge contre les Maladies neuro‐Musculaires (ABMM, Belgium). The authors also thank Michel Savels for his contribution to the
figure layout.
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