Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells

Cells and Culture conditions

The hESC lines HSF-1 (UC01) and HSF-6 (UC06) were cultured and maintained on irradiated mouse embryonic fibroblast cell lines as described earlier²⁵. The mESC line V6.5 was maintained on irradiated mouse embryonic fibroblasts in presence of LIF (Millipore). All work with hESCs was conducted with prior approval from the University of California Los Angeles Human Embryonic Stem Cell Research Oversight Committee. CCCP (carbonyl cyanide m-chloro phenyl hydrazone) (Sigma Aldrich), a chemical that uncouples oxidative phosphorylation from the electron transport chain was added to a final concentration of 2 μM to attenuate mitochondrial activity. This concentration of 2 μM was achieved after performing lethality analysis (LD50) with different concentrations of CCCP ranging from 1 μM to 12 μM. All the final concentrations of CCCP were prepared from a stock solution of 100mM in DMSO. Similar kind of solvent (DMSO) dilution was used for controls. For ATP and ROS assays self-renewing V6.5 and HSF1 cells were grown in absence of feeder cells on matrigel coated wells. Conditioned media (R&D Systems) was used to maintain the HSF1 cells in self-renewing state in a feeder free condition. The V6.5 cells were allowed to differentiate as an adherent culture in a feeder free condition with the withdrawal of LIF from the media. Differentiation of HSF1 cells was achieved by allowing them to grow as an adherent culture in absence of feeder cells and FGF2, and by replacing Knock-out (KO) Serum Replacement with Fetal Bovine Serum (FBS).

MitoTracker Red staining & Immunostaining

Live cells growing on cover-slips were incubated in the media containing MitoTracker Red (Molecular Probes) at a concentration of 250nM for 30 minutes, followed by three washes in the media and were fixed with 4% Formaldehyde in 1XPBS. The fixed cells were then permeabilized by treating with 0.2%TritonX 100 and immunostaining was performed following the protocol as described before ²⁵. The primary Antibodies used were anti OCT3/4 (1:100; Santa Cruz Biotechnology Inc), NANOG (1:200; Abcam) and Secondary Antibodies (1:200) were from Jackson Immunoresearch Laboratories.

ATP and Reactive Oxygen Species (ROS) Assays

Intracellular ATP level was measured by the ATP Bioluminescence Assay Kit HS II (Roche) according to supplier’s protocol. The luminescence intensity was measured by using a Glomax 96 Microplate Luminometer (Promega) and the luminescence readings were normalized by cell count. For measurement of ROS levels, cells were incubated in a medium containing 2 μM dihydroethidium (Molecular Probes) at 37°C in dark for 15 minutes. Cells were then washed with 1XPBS buffer and suspended in 1XPBScontaining 0.5% BSA. The fluorescence intensity of 30,000 cells was recorded with the help of a BD Biosciences LSR II (BD Biosciences).

Cell death Assay

Apoptosis was evaluated using 7-aminoactinomycin D (7-AAD; Calbiochem) according to the manufacturers instructions followed by flow cytometry.

Teratoma

ESCs were grown in presence or absence of CCCP under feeder free conditions to determine the ability of tumor formation by self-renewing or differentiating cells. The self-renewing cells were grown for four days and harvested on day 4 for injection. For the differentiating ESCs, the cells were grown in differentiation media (absence of LIF) for seven days and then harvested. The cells were injected into the testes of SCID mice following the protocol as described previously ²⁶. Surgery was performed following institutional approval for appropriate care and use of laboratory animals.

Analysis of Gene expression

RNA was extracted using the RNeasy Mini Kit (Qiagen), and reverse transcribed with Superscript II Reverse transcriptase (Invitrogen). Real time PCR was performed on the cDNA according to manufacturer’s protocol using Bio-Rad iQ iCycler with SYBR green as the fluorophore (Roche). For gene expression profiling of mouse and human stem cell samples, we used the Affymetrix Mouse Genome 430 2.0 Array and the Affymetrix human Genome U133plus2.0 array respectively. Two replicates were analyzed for each. Selection criteria for differential expression were performed as mentioned in the manuscript.

Dynamicity in mitochondrial morphology of self-renewing ESCs

Mitochondrial morphology was evaluated in self-renewing mouse and human ESCs by staining with a mitochondrial specific dye, Mitotracker Red. Consistent with previous reports, we found that both in self-renewing mouse (V6.5) and human (HSF1 and HSF6) ESCs mitochondria are distributed as perinuclear clusters that appear punctate and globular (Fig 1A–C″). Interestingly, similar mitochondrial morphology and distribution are observed even in human induced pluripotent cells (hiPS1; Fig. 1E, E′) derived by reprogramming dermal fibroblasts (Fig. 1D) with OCT4, SOX2, cMYC and KLF4 retroviruses ²⁷ suggesting that epigenetic reprogramming to generate hIPS cell lines is also associated with mitochondrial reprogramming. Although the common punctate and rudimentary appearance of mitochondria are seen in all undifferentiated ESCs, the amount of mitochondrial mass varies between independent lines of hESCs. For instance HSF6 cells appear to have more mitochondria than HSF1 cells (compare Fig 1B and C). Interestingly, regardless of the cell line, a modest increase in mitochondrial content associated with mitochondrial fusion to generate distinct tubules is seen during mitosis (Fig. 1G, H, & K, L). Following cytokinesis, mitochondria once again reorganize as perinuclear clusters (Fig. 1I, M). These cell cycle phase dependent changes in mitochondrial morphology indicate that mitochondria might play a specific functional role under self-renewing conditions.

Proliferation of ESCs under self renewing conditions depend on mitochondrial activity

To investigate the importance of mitochondrial oxidative phosphorylation in ESC self-renewal, mitochondrial activity was perturbed in self-renewing mouse (V6.5) and human (HSF1) ESCs, growing in absence of feeder cells, by culturing them in presence of 2 μM CCCP (carbonyl cyanide m-chloro phenyl hydrazone). We found that this treatment resulted in a 30% drop in cellular ATP levels in mouse ESCs (Fig 2A), and a 3–4 fold increase in the level of cellular Reactive Oxygen species (ROS) (Fig. 2B). As shown in Fig 2C, this attenuation of mitochondrial function affects the proliferation rate of mouse ESCs. However, no change in cell cycle phasing of CCCP treated mouse ESCs was apparent when compared with the untreated controls (Fig. 2D) indicating that while proliferation rate is reduced, this is not associated with a block in specific steps in cell cycle progression. Importantly, upon staining with 7ADD, a marker for apoptotic cells, no difference in the number of 7ADD positive cells was observed between mouse ESCs growing in presence or absence of CCCP (Fig 2E). Furthermore, no difference in the proportion of senesced cells as determined by negative Ki67 staining was observed under self-renewing conditions (Fig. 2F–G′). Evaluation of the human ESC line, HSF1 maintained in the presence of 2 μM CCCP, revealed that in a manner similar to the mouse V6.5 cells these cells demonstrate changes in the cellular ATP (Fig. 2H) and ROS levels (Fig. 2I) and proliferate slowly (Fig. 2J). Collectively, these results clearly suggest that the proliferation of self-renewing ESCs is dependent on mitochondrial function.

Pluripotency of self-renewing ESCs is not compromised upon blocking mitochondrial function

One of the fundamental properties of undifferentiated ESCs is to maintain a pluripotent state and therefore retain the capacity to generate endodermal, mesodermal, ectodermal and germ cell derivatives as they differentiate. In an attempt to understand the relationship between oxidative phosphorylation and pluripotency, we studied the mRNA levels of pluripotent transcription factors, OCT4, NANOG and SOX2 using real time RT-PCR in both mouse (Fig 3A) and human (Fig. 3B) ESCs grown in presence or absence of CCCP. We found that the expression of OCT4, NANOG and SOX2 transcripts were increased by at least two fold in both mouse and human ESCs treated with CCCP compared to their untreated controls. In order to evaluate the pluripotency of CCCP treated ESCs, we transplanted the treated and control ESCs into the testicles of adult SCID mice (n=4). Tumor formation was achieved in 100% of transplants from wild type and CCCP treated ESCs. Histological sections of the tumors revealed that CCCP treated ESCs generated teratomas, which were comparable to control, treated ESCs and showed evidence of differentiation into all three embryonic lineages (Fig. 3C–F).

In order to confirm that CCCP treated mouse ESCs have attenuated mitochondrial function, we examined glycolytic flux (Fig. 3G) and the rate of oxygen consumption (Fig. 3H) in treated verses control ESCs cultured under self-renewing conditions. These results demonstrate that in the presence of CCCP, undifferentiated ESCs exhibit reduced consumption of oxygen due to the uncoupling of the electron transport chain and a concomitant increase in glycolytic flux. Taken together, these results demonstrate that blocking oxidative phosphorylation with CCCP in undifferentiated ESCs increases glycolytic metabolism and reduces proliferation rate which are associated with increased transcriptional of the three critical pluripotency transcription factors Oct4, Nanog and Sox2.

Differentiation of ESCs is associated with changes in mitochondrial morphology

Previous studies have demonstrated that compared to the punctate globular mitochondria, as found in undifferentiated ESCs, differentiation results in the generation of an extensive network of branched mitochondria ^{13, 15}. However correlating the kinetics of network formation with loss of pluripotent gene expression has never been performed. To achieve this, we evaluated the temporal changes in mitochondrial morphology with respect to the loss of OCT4 (Fig. 4A–D′) and NANOG (Fig. 4E–H′) in differentiating HSF1 cells. Differentiation was performed as an adherent culture in the absence of FGF2 and feeder cells, and by substituting Knock-out Serum Replacement with Fetal Bovine Serum. As shown in Fig. 4B, B′, the formation of mitochondrial network was initiated on day 3 of differentiation while the cells maintained high levels of OCT4 protein. On subsequent days of differentiation as OCT4 was lost, the cells acquired an extensive network of mitochondrial branching (Fig. 2C–D′). Identical temporal profile of mitochondrial branching was seen with respect to loss of NANOG expression in differentiating HSF1 cells (Fig. 4E–H′). Consistent with the formation of extensive mitochondrial network, differentiating HSF1 cells exhibited a gradual increase in cellular ATP and ROS levels. Our results, therefore, demonstrate that at a single cell level, mitochondrial branching precedes the loss of pluripotency markers and thereby suggests that establishing appropriate metabolic status of a cell is one of the earliest fate changes that occur when differentiation is initiated.

Proliferation and Early Differentiation is affected when differentiation is conducted under conditions that attenuate mitochondrial activity

To examine the importance of oxidative phosphorylation during early cell fate lineage commitment, we blocked oxidative phosphorylation in differentiating ESCs for seven days by growing them in presence of CCCP. This treatment dropped cellular ATP levels by 40% in both mouse and human ESCs (Fig. 5A). Furthermore, we determined that ROS levels were initially increased relative to control in the first few days after CCCP treatment, however, after seven days the level of ROS was normalized to wild type (data not shown) presumably due to the high levels of antioxidants produced by differentiating ESCs to keep ROS in balance ¹⁵. Staining the differentiated cells with Mitotracker Red revealed that, unlike controls, CCCP treated mouse and human differentiated ESCs exhibit an apparent discontinuous network (Fig. 5B–G), likely due to a block in mitochondrial fusion. Interestingly as observed for self-renewing ESCs, CCCP treatment affected the proliferation rate of differentiating mouse and human ESCs (Fig. 5H, I). Although the rate of proliferation were comparable for the first three days of differentiation, on subsequent days the CCCP treated differentiating cells demonstrated significantly reduced proliferation rates suggesting that oxidative phosphorylation is also required for proliferation during the process of differentiation.

Differentiation of ESCs is characterized by the loss pluripotency, and a concomitant turning on of transcriptional programs required for differentiation. In an attempt to understand the contribution of mitochondrial activity in the process of differentiation we induced differentiation in the presence of CCCP. Our results show that CCCP treatment during differentiation had no effect on the repression of Oct4, Sox2 and Nanog expression in either mouse or human ESCs (Fig. 5J & K).

To identify transcriptional consequences of CCCP treatment during differentiation, we performed genome wide expression profiling comparing ESCs differentiated in CCCP for 7 or 10 days (mouse and human respectively) to that of untreated control cells. Using the GeneChip Mouse Genome 430 2.0 Array, we found 1200 genes differentially expressed by a factor of 2 or more on the basis of a t test p value of 0.001, in differentiated mouse ESCs treated with CCCP. Gene ontology (GO) based classification revealed that the differentially expressed genes are associated with early development and differentiation (Fig. 6A), with a notable decrease in Hox gene expression (Fig. 6C) in differentiating mouse ESCs treated with CCCP. Using the Affymetrix human chip, U133plus2.0 array microarrays we compared differentiated human ESCs cultured in the presence or absence of CCCP. Applying the same set of criteria we found that 410 genes were differentially expressed between control and CCCP treated differentiating hESCs cells. GO analysis revealed that CCCP treated differentiating hESCs also exhibited differentially expressed genes associated with development and differentiation (Fig. 6B). Furthermore, in a manner similar to mouse ESCs, differentiated human ESCs demonstrated a significant decrease in the expression of HOX genes when differentiated in the presence of CCCP (Fig. 6D). Together, these results clearly demonstrate that in differentiating ESCs normal mitochondrial activity is critical for the onset of differentiation.

Differentiating ESCs attenuated for mitochondrial activity cause teratoma

Given that repression of Oct4, Nanog and Sox2 mRNA were unchanged relative to controls, but execution of transcriptional programs associated with differentiation were compromised in CCCP treated differentiating ESCs, we next evaluated whether differentiating in the presence of CCCP promotes the persistence of cells that are still capable of tumor formation. To achieve this we transplanted V6.5 cells differentiated in the presence or absence of CCCP for seven days, into the testicles of adult SCID mice. Transplantation of untreated differentiated cells resulted in zero tumor formation 6 weeks after transplant²⁶. In contrast, cells differentiated in the presence of CCCP resulted in tumor formation in 100% of transplants (n=4). Histological analysis of these tumors showed teratoma formation with differentiation into cell types representing the three embryonic lineages (Fig. 6E–G). These results indicate that CCCP treatment during the process of differentiation promotes the persistence of cells with tumorigenic potential when transplanted into SCID mice.