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Mitochondrial Function Controls Proliferation and Early Differentiation Potential of Embryonic Stem Cells

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From the March 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Mitochondrial function is understood to play a key role in the ageing process and mitochondrial dysfunction underlies the pathophysiology of various diseases. Whilst much attention has focused on the role of the genetic and epigenetic state on cell function and differentiation in stem cells, little work thus far has addressed the contribution of cell metabolism in stem cell function and activity. New results reported in the March edition of Stem Cells by Mandal et al.1 from the University of California and Indian Institute of Science Education and Research, now begin to reveal the relationship between the mitochondria and stem cell proliferation, differentiation and tumorigenesis.

Under self-renewing conditions human embryonic stem cells (hESCs) typically show a high nuclear-to-cytoplasmic ratio with few mitochondria containing poorly developed cristae, limited oxidative capacity and demonstrate the ability to function efficiently under anaerobic (low oxygen) conditions. Upon differentiation the mitochondrial compliment increases, numerous mitochondrial cristae develop and oxidative phosphorylation increases. This indicates that glycolytic mechanisms, and not oxidative phosphorylation, predominate under proliferative conditions therefore Mandal et al.1 set out to evaluate any potential roles for mitochondrial oxidative phosphorylation in hESC self-renewal. The authors attenuated mitochondrial function in proliferative and differentiating stem cells by chemical means, using controlled doses of Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP), which allows protons to cross lipid bilayers. This acts to depolarise the inner mitochondrial membrane, hence uncoupling oxidative phosphorylation from the electron transport chain. Staining mitochondria with MitoTracker red, Mandal et al.1 noted that under proliferative conditions in mouse ESC (mESC), hESC and human dermal-derived induced pluripotent stem cells (hiPSC), mitochondria exist as perinuclear clusters but the amount of mitochondrial mass between individual cell lines varies. Perturbing mitochondrial activity with CCCP in mESC and hESC caused a 40% reduction in cellular ATP levels and a transient increase in cellular reactive oxygen species (ROS), alongside a reduction in the rate of proliferation. Similar results were observed in hiPSCs, indicative that oxidative phosphorylation plays a role in stem cell self-renewal. Treated cells also showed reduced oxygen consumption and increased glycolytic flux. Interestingly, CCCP treatment caused increased transcription of OCT4, SOX2 and NANOG in ESCs but was not observed to cause any differences in teratoma formation when compared with untreated controls with both cell types generating teratomas which displayed differentiated cell types from all three germ layers.

The authors then documented the changes in mitochondrial morphology that accompany the downregulation of pluripotency markers upon differentiation. Formation of a mitochondrial network was observed after three days of differentiation while OCT4 was still high, with a more complex network of mitochondrial branching developing as OCT4 and NANOG levels decreased, alongside increased cellular ATP and a transient increase in ROS levels. As mitochondrial branching precedes the loss of pluripotency markers, the authors suggest that the establishment of the metabolic status of the cell is one of the earliest fate changes prior to the initiation of differentiation.

Although CCCP treatment did not affect teratoma formation in vivo, in vitro differentiation of stem cells with perturbed mitochondria revealed a discontinuous cytoplasmic mitochondrial network, as though the normal processes of mitochondrial fusion had been repressed, as well as a reduction in cellular proliferation. Genome wide expression profiling of differentiating mESC revealed that CCCP treatment elicited differential gene expression in 1200 genes associated with early development and differentiation, with a notable decrease in Hox genes. Affymetrix profiling revealed 410 differentially expressed genes in CCCP-treated hESC, again with a notable reduction in HOX genes. This implies that normal mitochondrial activity is essential for various transcriptional events occurring prior to and during the differentiation process.

Given that the downregulation of pluripotency genes upon differentiation between control and CCCP-treated cells was similar yet downstream transcriptional events were affected, the authors questioned whether mitochondrial function perturbation might affect the tumorigenic potential of the cells. Likely to be the most revelationary result of the paper, Mandal et al.1 report that while control ESCs differentiated for seven days in vitro showed no tumor formation in vivo, those with dysfunctional mitochondria resulted in tumor formation in all cases, forming teratomas containing cells from all three germ layers. Thus, attenuation of mitochondrial function during differentiation enabled the persistence of cells with tumorigenic potential. Whether this arose from a small proportion of stem cells persisting in the differentiated population or the reversion of differentiating cells with attenuated mitochondria to a pluripotent state was not investigated.

The observed link between normal mitochondrial function, early lineage specification and tumor formation in this paper is of great interest and may have important implications for the choice of stem cells that reach the clinic for various stem cell therapies, not only for safety considerations, but for differentiation capability. Whilst apoptosis and cell senescence remained unaffected as far as these factors were investigated in this study, mitochondrial perturbation did cause a proportional slowing of all phases of the cell cycle and reduced the rate of ESC self-renewal, a surprising result given the subsequent tumorigenic potential of differentiating cells under mitochondrial perturbation, although this might be linked to the initial increase in transcription of pluripotency markers under CCCP treatment. The similarity of mitochondrial localisation and morphology between ESCs and hiPSCs is also an interesting result given the recent barrage of reported differences between these pluripotent cell types, and indicates that both at least rely on similar cellular metabolism during self-renewal.

 

Original Article

1. Mandal et al. Mitochondrial Function Controls Proliferation and Early Differentiation Potential of Embryonic Stem Cells. Stem Cells. March 2011.