Background: To date, many attempts are employed to increase the regenerative potential of stem cells. In this study, we evaluated the hypothesis whether an autophagy modulation could induce/reduce CD146+ cells differentiation into mature pericyte, endothelial and cardiomyocyte lineage. Methods In this study, CD146+ cells were enriched from human bone marrow aspirates and trans-differentiated into mature EC, PC and CM after exposure to autophagy stimulator (50 µM Met)/inhibitor (15 µM HCQ). The protein levels of autophagy proteins were monitored by western blotting. NO content was measured using the Griess assay. Using real-time PCR assay and western blotting, we monitored the lineage protein and gene levels. The fatty acid change was determined by gas chromatography. Pro-inflammatory cytokine and angiocrine factors were measured by ELISA. The exosome secretion capacity was assessed by AChE activity and real-time PCR assay. Result Data revealed the modulation of autophagy factors, Beclin-1, P62 and LC3 II/I ratio in differentiating CD146+ cells after exposure to Met and HCQ (p<0.05). The inhibition of autophagy increased released NO content and decreased intracellular NO content compared to the Met-treated cells (p<0.05). Real-time PCR analysis showed that the treatment of CD146+ cells with autophagy modulators altered the expression of VE-cadherin, cTnI and α-SMA. Our data demonstrated that the stimulation of autophagy signaling in CD146+ cells with Met increased the expression of VE-cadherin, α-SMA, and cTnI compared to HCQ-treated cells (p<0.05) while western blotting revealed the protein synthesis of all lineage-specific proteins in under the stimulation and inhibition of autophagy. Fatty acid profile analysis revealed the increase of unsaturated fatty acids after exposure to HCQ (p<0.05). None statistically significant differences were found in the levels of Tie-1, Tie-2, VEGFR-1 and VEGFR-2 after autophagy modulation. The treatment of cells with HCQ increased the levels of TNF-α and IL-6 compared to the Met-treated cells. Data revealed the increase of exosome biogenesis and secretion to the supernatant in cells treated with HCQ compared to the Met groups (p<0.05).ConclusionsIn summary, autophagy modulation could be altered differentiation potency of CD146+ cells and could be novel and applicable cardiac cell therapy in the cardiac regeneration field.

Background: Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. This enzyme can orchestrate posttranslational modification of proteins involved in the development and differentiation of cells through the fatty acid acylation process. In this study, we evaluated whether a small molecule modulating unsaturated fatty acids influences early endodermal differentiation of induced pluripotent stem cells, using biochemical methods and immunostaining.Methods: The hiPSCs were cultured in an endoderm-inducing medium containing activin A and low defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The yield of three germ layers endoderm, mesoderm, and ectoderm, and the cell cycle analysis were assessed using flow cytometry. The expression of endoderm and pluripotency markers, as well as the expression of Wnt signaling pathway proteins, were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction.Results: The population of cells showing endoderm features was decreased at the end of differentiation when SCD1 was inhibited on the first day. Moreover, early SCD1 inhibition preserved hiPSCs properties without a shift toward mesoderm or ectoderm. Treatment of cells with SCD1 inhibitor only on the first day decreased the β-catenin gene expression and intensity of fluorescent emission in the click chemistry. These effects were effectively rescued by cotreatment with oleate. Late treatment at two subsequent days of endoderm induction induced no significant effect on endoderm-specific markers and fluorescent intensity. Reproducible results were also obtained with a human embryonic stem cell line. Conclusion: The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring maintenance to hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in endoderm commitment of pluripotent stem cells may be eminent in disorders of endoderm-derived organs and dysregulated metabolism.