Figure 2
CD34 positive cells selected from PR-SMCs can differentiate into a more enriched endothelial-like cell population. (a) Schematic protocol of selecting CD34 positive cells from PR-SMCs and further differentiation towards the endothelial lineage. (b) CD34 positive cells-derived endothelial cells (SMC-CD34-ECs) displayed an endothelial-like monolayer compared to SMCs. (Scale Bar: 100 μm) (c) Real-time PCR result showed a strong induction of endothelial markers including KDR, CD31, CD144, eNOS, vWF and a strong suppression of SMC markers α-SMA, SM22α, Calponin and SM-MHC. (*p < 0.05, ***p < 0.001 by Student’s t test, n = 4) (d) Hierarchical clustering analysis of global gene expression from RNA-Seq of SMCs, PR-SMCs, SMC-CD34-ECs and HUVECs showed a shift of the overall gene expression towards ECs. (e,f) Heat map of EC and SMC enriched gene expression changes among SMC, SMC-ECs and SMC-CD34-ECs based on the RNA-Seq results. Colour bar indicates gene expression in scale. (g) Immunofluorescence staining of SMC-CD34-ECs revealed the typical junctional staining pattern of the endothelial markers CD31 and CD144. (Scale bar: 25 μm) (h) Flow cytometry analysis evaluated the increase of the progenitor marker CD34 and endothelial marker CD31 in SMC-CD34-ECs compared to SMCs.
