Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4

The molecular mechanisms underlying the regulation of pluripotency by cellular metabolism in human embryonic stem cells (hESCs) are not fully understood. We found that high levels of glutamine metabolism are essential to prevent degradation of OCT4, a key transcription factor regulating hESC pluripotency. Glutamine withdrawal depletes the endogenous antioxidant glutathione (GSH), which results in the oxidation of OCT4 cysteine residues required for its DNA binding and enhanced OCT4 degradation. The emergence of the OCT4lo cell population following glutamine withdrawal did not result in greater propensity for cell death. Instead, glutamine withdrawal during vascular differentiation of hESCs generated cells with greater angiogenic capacity, thus indicating that modulating glutamine metabolism enhances the differentiation and functional maturation of cells. These findings demonstrate that the pluripotency transcription factor OCT4 can serve as a metabolic-redox sensor in hESCs and that metabolic cues can act in concert with growth factor signaling to orchestrate stem cell differentiation

Mitochondrial redox status changes with hMSCs adipogenic differentiation.

<p><b>A)</b> Undifferentiated and 7 day adipogenic differentiated MSCs were transfected with a redox-sensitive GFP construct (roGFP) targeted to the mitochondria. By using different excitation wavelengths (400 and 490 nm) and measuring emission at 535 nm, the redox status of cells was assessed (a higher 400/490 ratio corresponds to a more oxidized mitochondrial matrix). Ratios are represented in the form of a heat map, with reduced mitochondria shown in blue and oxidized mitochondria in red. <b>B)</b> Quantification of the roGFP data. Mitochondrial redox state is reduced after adipogenic differentiation. As a positive control, cells were also treated with 100 µM H₂O₂ to induce a completely oxidized state. <b>C)</b> Immunoblotting indicates that catalase and mitochondrial superoxide dismutase (SOD2) protein levels increased during differentiation, while cytoplasmic superoxide dismutase (SOD1) levels were not significantly affected by differentiation (n = 3 for each group).</p

TFAM knockdown inhibits adipogenic differentiation of hMSCs.

<p><b>A)</b> Immunofluorescent staining for TFAM, the key regulator of mitochondrial transcription, showed that TFAM is upregulated in hMSCs undergoing adipogenic differentiation. <b>B)</b> High dose siTFAM can significantly lower TFAM expression levels (n = 4 for each group). <b>C)</b> Knockdown of TFAM inhibits the differentiation process as confirmed by lower adiponectin mRNA levels (n = 4 for each group). <b>D)</b> Lowering TFAM results in lower expression of the mitochondrial gene MtND2, while the nuclear gene cytochrome C (CytC) is not affected, confirming the specificity of siRNA treatment (n = 3 for each group).</p

Mitochondrial Respiration Regulates Adipogenic Differentiation of Human Mesenchymal Stem Cells

<div><p>Human mesenchymal stem cells (MSCs) are adult multipotent stem cells which can be isolated from bone marrow, adipose tissue as well as other tissues and have the capacity to differentiate into a variety of mesenchymal cell types such as adipocytes, osteoblasts and chondrocytes. Differentiation of stem cells into mature cell types is guided by growth factors and hormones, but recent studies suggest that metabolic shifts occur during differentiation and can modulate the differentiation process. We therefore investigated mitochondrial biogenesis, mitochondrial respiration and the mitochondrial membrane potential during adipogenic differentiation of human MSCs. In addition, we inhibited mitochondrial function to assess its effects on adipogenic differentiation. Our data show that mitochondrial biogenesis and oxygen consumption increase markedly during adipogenic differentiation, and that reducing mitochondrial respiration by hypoxia or by inhibition of the mitochondrial electron transport chain significantly suppresses adipogenic differentiation. Furthermore, we used a novel approach to suppress mitochondrial activity using a specific siRNA-based knockdown of the mitochondrial transcription factor A (TFAM), which also resulted in an inhibition of adipogenic differentiation. Taken together, our data demonstrates that increased mitochondrial activity is a prerequisite for MSC differentiation into adipocytes. These findings suggest that metabolic modulation of adult stem cells can maintain stem cell pluripotency or direct adult stem cell differentiation.</p></div

Mitochondrial membrane potential changed with hMSC adipogenic differentiation.

<p><b>A)</b> JC-1 staining was used for the measurement of mitochondrial membrane potential. The ratio of red/green (and thus polarization) decreased with adipogenic differentiation. Note that not all cells were fully differentiated even after 21 days. Asterisks highlight the smaller, undifferentiated cells, while the arrow points at a larger and well-differentiated cell that contains multiple lipid droplets. Upon differentiation, mitochondrial depolarization (green color) was clearly present. <b>B)</b> Real-time RT-PCR data showed increased expression of PGC-1α and of the 3 uncoupling proteins (UCP1, 2, 3) following adipogenic differentiation (n = 5 for each group).</p

Adipogenic differentiation enhances mitochondrial oxidation in hMSCs.

<p><b>A)</b> Bone marrow-derived hMSCs underwent adipogenic differentiation using 500 nM Isobutylmethylxanthine, 1 µM Dexamethasone, 50 µM Indomethacin and 5 µg/ml Insulin. Oil Red O staining was used to confirm the adipogenic differentiation of hMSCs at day 21 (n = 4 and representative pictures are shown). <b>B)</b> Real-time RT-PCR confirmed the upregulation of the adipogenic differentiation marker adiponectin (n = 5 for each group). <b>C)</b> Oxygen consumption rate (OCR) increases gradually during adipogenic differentiation. Furthermore, the maximal OCR as elicited by treatment with the mitochondrial uncoupler FCCP (2 µM) is also increased during adipogenic differentiation (n≥5 for each group). <b>D)</b> Lactate production was decreased after adipogenic differentiation, indicating decreased glycolysis upon differentiation (n = 9 for each group). <b>E)</b> Cellular ATP content normalized to total cellular protein decreased gradually during 7 days of adipogenic differentiation (n = 5 for each group).</p

Mitochondrial biogenesis increased with adipogenic differentiation of hMSCs.

<p><b>A)</b> Adipogenic differentiation was associated with a marked increase in mitochondrial mass, as demonstrated by increased MitoTracker Green staining. <b>B)</b> Flow cytometry measurement of MitoTracker Green staining confirmed the increase of mitochondrial mass as the mean fluorescence intensity is doubled after 7 days of adipogenic differentiation (n = 3 for each group). <b>C)</b> The protein levels of the mitochondrial outer membrane protein TOM20, a reliable marker of mitochondrial mass, showed a marked increase after adipogenic differentiation.</p

Inhibition of mitochondrial oxidation prevents adipogenic differentiation of hMSCs.

<p><b>A)</b> 100 nM rotenone decreased the baseline oxygen consumption rate (OCR) and maximal respiration capacity (induced by FCCP treatment) in hMSCs (n = 7 for each group). <b>B)</b> Oil Red O staining showed that chronic treatment with 100 nM Rotenone inhibited adipogenic differentiation with a significant decrease in the percentage of surface area stained with Oil Red O (n = 5 for DMSO control and n = 4 for 100 nM Rotenone treatment). <b>C)</b> Real-time RT-PCR data confirm the inhibition on hMSCs adipogenic differentiation by rotenone as adiponectin levels are significantly lower after rotenone treatment (n = 3 for each group). <b>D)</b> Importantly, chronic treatment with 100 nM rotenone for 7 days during adipogenic differentiation did not result in ATP depletion, thus suggesting that the concentration of rotenone used in our studies was not toxic (n = 3 for each group).</p

Bioenergetic shifts during transitions between stem cell states

Two defining characteristics of stem cells are their multilineage differentiation potential (multipotency or pluripotency) and their capacity for self-renewal. Growth factors are well-established regulators of stem cell differentiation and self renewal, but less is known about the influence of the metabolic state on stem cell function. Recent studies investigating cellular metabolism during the differentiation of adult stem cells, human embryonic stem cells (ESCs), and induced pluripotent stem cells have demonstrated that activation of specific metabolic pathways depends on the type of stem cells as well as the lineage cells are differentiating into and that these metabolic pathways can influence the differentiation process. However, some common patterns have emerged, suggesting that undifferentiated stem cells primarily rely on glycolysis to meet energy demands. Our own data indicate that undifferentiated ESCs not only exhibit a low mitochondrial membrane potential but also express high levels of the mitochondrial uncoupling protein 2 and of glutamine metabolism regulators when compared with differentiated cells. More importantly, interventions that target stem cell metabolism are able to either prevent or enhance differentiation. These findings suggest that the metabolic state of stem cells is not just a marker of their differentiation status but also plays an active role in regulating stem cell function. Regulatory metabolic pathways in stem cells may thus serve as important checkpoints that can be modulated to direct the regenerative capacity of stem cells

SLIT3–ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo

Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called “pericytic” MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, “non-pericytic” MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications