SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells

BACKGROUND: The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. METHODS: CD34+ progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34+ cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. RESULTS: Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34+ progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. CONCLUSIONS: Dedifferentiation of fibroblasts to intermediate CD34+ progenitors gives rise to endothelial cells and erythroblasts in a SOX17-dependent manner. These findings identify the intermediate CD34+ progenitor state as a critical bifurcation point, which can be tuned to generate functional blood vessels or erythrocytes and salvage ischemic tissue

BNip3 regulates mitochondrial function and lipid metabolism in the liver

BNip3 localizes to the outer mitochondrial membrane, where it functions in mitophagy and mitochondrial dynamics. While the BNip3 protein is constitutively expressed in adult liver from fed mice, we have shown that its expression is superinduced by fasting of mice, consistent with a role in responses to nutrient deprivation. Loss of BNip3 resulted in increased lipid synthesis in the liver that was associated with elevated ATP levels, reduced AMP-regulated kinase (AMPK) activity, and increased expression of lipogenic enzymes. Conversely, there was reduced β-oxidation of fatty acids in BNip3 null liver and also defective glucose output under fasting conditions. These metabolic defects in BNip3 null liver were linked to increased mitochondrial mass and increased hepatocellular respiration in the presence of glucose. However, despite elevated mitochondrial mass, an increased proportion of mitochondria exhibited loss of mitochondrial membrane potential, abnormal structure, and reduced oxygen consumption. Elevated reactive oxygen species, inflammation, and features of steatohepatitis were also observed in the livers of BNip3 null mice. These results identify a role for BNip3 in limiting mitochondrial mass and maintaining mitochondrial integrity in the liver that has consequences for lipid metabolism and disease

Gender-Specific Modulation of the Response to Arterial Injury by Soluble Guanylate Cyclase α1

Objective: Soluble guanylate cyclase (sGC), a heterodimer composed of α and β subunits, synthesizes cGMP in response to nitric oxide (NO). NO modulates vascular tone and structure but the relative contributions of cGMP-dependent versus cGMP-independent mechanisms remain uncertain. We studied the response to vascular injury in male (M) and female (F) mice with targeted deletion of exon 6 of the sGCα1 subunit (sGCα1-/-), resulting in a non-functional heterodimer. Methods: We measured aortic cGMP levels and mRNA transcripts encoding sGC α1, α2, and β1 subunits in wild type (WT) and sGCa1-/- mice. To study the response to vascular injury, BrdU-incorporation and neointima formation (maximum intima to media (I/M) ratio) were determined 5 and 28 days after carotid artery ligation, respectively. Results: Aortic cGMP levels were 4-fold higher in F than in M mice in both genotypes, and, within each gender, 4-fold higher in WT than in sGCa1-/-. In contrast, sGCα1, sGCα2, and sGCβ1 mRNA expression did not differ between groups. 3H-thymidine incorporation in cultured sGCa1-/- smooth muscle cells (SMC) was 27%±12% lower than in WT SMC and BrdU-incorporation in carotid arteries 5 days after ligation was significantly less in sGCa1-/- M than in WT M. Neointima area and I/M 28 days after ligation were 65% and 62% lower in sGCa1-/- M than in WT M mice (p<0,05 for both) but were not different in F mice. Conclusion: Functional deletion of sGCa1 resulted in reduced cGMP levels in male sGCa1-/- mice and a gender-specific effect on the adaptive response to vascular injury

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 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 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 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