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

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

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

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 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

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

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

Flk1+ and VE-Cadherin+ Endothelial Cells Derived from iPSCs Recapitulates Vascular Development during Differentiation and Display Similar Angiogenic Potential as ESC-Derived Cells

<div><p>Rationale</p><p>Induced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1⁺ and Vascular Endothelial (VE)-cadherin⁺ ECs derived identically from mouse (m)ES and miPS cells. </p> <p>Methods and Results</p><p>Naive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1⁺ VE-cadherin⁺ cells from both mES and miPS cells. Emergence of Flk1⁺VE-cadherin⁺ cells entailed expression of the vascular developmental transcription factor <i>Er71</i>, which bound identically to <i>Flk1, VE-cadherin</i>, and <i>CD31</i> promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures <i>in</i><i>vitro</i> and incorporated into CD31⁺ neovessels in matrigel plugs implanted in nude mice <i>in</i><i>vivo</i>.</p> <p>Conclusion</p><p>Thus, iPS cell-derived Flk1⁺VE-cadherin⁺ cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31⁺ neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.</p> </div

miPS derived ECs in the Matrigel plug are not mobilized to subsequent areas of injury.

<p>Mice were subjected to hindlimb ischemia 7 days after injection of Matrigel containing either no cells, mouse ECs or iPS cell-derived ECs. Cells were transduced with mCherry lentivirus particles before mixing with Matrigel. Ischemic tibialis anterior (TA) muscles were harvested 14 days after the surgery (21 days after Matrigel implantation). Cross sections of frozen tissues were observed under laser confocal microscopy. Images acquired with appropriate filter set for mCherry detection show fluorescent signals (20X objective) (A). Quantification demonstrated no appreciable difference in the numbers of fluorescent cells between the groups, suggesting that the obtained signals were likely due to autofluorescence and not due to a mobilization of the Matrigel-engrafted iPS-ECs (B). Fluorescence spectrum images were acquired by excitation with 405 and 562 nm and detection wave length with 470 nm and from 577 to 684 by 9-10 nm steps. The spectral analysis confirmed that the fluorescent signals from the hindlimbs of mice containing Matrigel plugs with mCherry labeled cells and those containing no cells were overlapping. Therefore, the fluorescent signals seen in (A) were indicative of autofluorescence of endogenous cells and did not demonstrate mobilization of iPS-ECs into the ischemic TA muscle (C). Experiments were repeated 3 times.</p

Induction of Flk1⁺VE-cadherin⁺ vascular EC progenies from iPS and ES cells.

<p>Timeline of emergence of Flk1⁺VE-cadherin⁺ vascular ECs (<b>A</b>). Undifferentiated mES (J1 line) or miPS (iMZ-21) cells were cultured for 5 d in IV Col-coated dishes in media containing BMP4, bFGF, and VEGF¹⁶⁵ to induce generation of vascular EC progenies. (B&C) Representative phase contrast microscopy of mES cell-derived adherent vascular progenies after d 5 in culture at indicated magnifications (B&C). Representative phase contrast microscopy of miPS cell-derived vascular progenies after day 5 in culture at the indicated magnifications (E&F). FACS profile of the emergence of Flk1⁺VE-cadherin⁺ vascular progenies (D&G). All experiments were repeated >5 times. Data indicate the mean±S.E.M. n=5.</p