Generation of vascular smooth muscle cells from human pluripotent stem cells via the activation of Wnt and BMP signaling

Abstract

Objective To establish an optimized protocol for rapid and efficient differentiation of human pluripotent stem cell into vascular smooth muscle progenitor cells with serum-free and chemically defined medium. Methods Monolayer H1/H9 embryo stem cells(ESC) and induced pluripotent stem cells(iPSC) were cultured in serum-free and chemically defined medium, and divided into two groups according to the supplemented growth factors in the first two days. A group: added bone morphogenetic protein 4(BMP4) and Activin A; B group: supplemented BMP4, Activin A and CHIR99021. CD31+ , CD34+ vascular progenitor cells were sorted by flow cytometry at day 9, and then directly differentiated into vascular smooth muscle cells with PDGF-BB. The differentiated cells were characterized by immunofluorescent staining and real-time quantitative PCR. Results Flow cytometric analyses demonstrated the percentage of CD31+ , CD34+ cells sorted from the differentiated cell population in A group is significantly higher than that in B group(38.8%-51.4% vs. 11.7%-22.5%. P<0.05). After in vitro direct differentiation, all differentiated cells stained positively and had gene expressions of myogenic marker, such as SM22-α, SMA-α and Calponin. Conclusion Simultaneous activation of the BMP signaling and Wnt signaling can rapidly and efficiently induced the hPSCs differentiation into vascular smooth muscle cells. It holds a promise to provide a population of clinical-grade vascular smooth muscle cells for cell therapy. Key words: Multipotent stem cells; Myocytes, smooth muscle; Induction; Cell differentiation