Figure 1
Conversion of human SMCs toward endothelial-like cells through short term dedifferentiation using reprogramming factors. (a) Schematic protocol of the conversion of SMCs towards endothelial-like cells. SMCs were transfected with lentivirus vector containing the four reprogramming factors Oct4, Sox2, Klf4, c-Myc and then maintained in the reprogramming media for 4 days. Partially reprogrammed SMCs (PR-SMCs) were then differentiated towards endothelial lineage by culturing with endothelial-inductive condition. (b) Real-time PCR showed the induction of the four reprogramming factors at the mRNA level of PR-SMCs. Control group refers to the SMCs transfected with empty lentivirus vector and maintained under the same reprogramming conditions. (***p < 0.001 by Student’s t test, n = 4) (c) Real-time PCR showed an upregulation of progenitor marker CD34 in PR-SMCs. (***p < 0.001 by Student’s t test, n = 4) (d) Flow cytometry analysis of CD34 positive cells in PR-SMC population. (*p < 0.05 by Student’s t test, n = 3) (e) Real-time PCR results revealed an overall induction of the endothelial markers CD31, CD144, eNOS, vWF and Claudin5 (CLDN5) at the mRNA level in SMC-derived endothelial like cells (SMC-ECs). (*p < 0.05, ***p < 0.001 by Student’s t test, n = 5). Control group refers to the SMC transfected with empty lentiviral vector that underwent the same reprogramming and differentiation protocol. (f) Representative images of the immunofluorescence staining for CD31 of SMC-ECs. (Scale Bar: 25 μm) (g) Western blot analysis showed the expression of CD31 and CD144 at the protein level of SMC-ECs. (h) By immunofluorescence staining, white arrows indicated the cells that started to express vWF were the cells that lost the typical filamentous αSMA expression in the SMC-ECs population. (Scale bar: 25 μm) (i) Flow cytometry analysis exhibited increased CD31, CD34 expression in SMC-ECs compared to control group. Western blots were cropped for clarity. Examples of uncropped blots are found in Supplementary Figure 15.
