2023, Vol. 27 ›› Issue (10): 1553-1559

Establishment and identification of an efficient protocol for differentiation of endothelial cells from human induced pluripotent stem cells

Huang Wenjun1, 2, 3, 4, 5, Wang Jie1, 2, 3, 4, 5, Zhou Yafei1, 2, 3, 4, 5, Li Huan1, 2, 3, 4, 5, Jiang Congshan1, 2, 3, 4, 5, Zhang Yanmin1, 2, 3, 4, 5, Zhou Rui1, 2, 3, 4, 5   

Abstract: BACKGROUND: Similar to the human induced embryonic stem cells, human induced pluripotent stem cell is a kind of pluripotent stem cells. By virtue of the differentiation potential of three germ layers and self-renewal ability, it could serve as a means to generate a scalable source of cells for therapeutic applications.
OBJECTIVE: To establish a protocol for the directed differentiation of human induced pluripotent stem cells into functional endothelial cells.
METHODS:   The authenticity of human induced pluripotent stem cells was verified by the immunofluorescence method. Human induced pluripotent stem cells were then differentiated into cardiac mesoderm, endothelial progenitor cells and finally functional endothelial cells through the manipulation of signaling pathways using recombinant transcription factors and small molecule compounds by adding activin A, GSK pathway inhibitor CHIR99021/bone morphogenetic protein 4, fibroblast growth factor/vascular endothelial growth factor/bone morphogenetic protein 4, basic fibroblast growth factor/vascular endothelial growth factor/CHIR99021 on days 0, 1, 2, and 5 sequentially. The induced endothelial cells were characterized by the morphology and the expression of endothelial cells-specific markers using brightfield microscope, real-time quantitative PCR and immunofluorescence method. Moreover, the tube formation assay was performed to test the angiogenetic ability of induced endothelial cells.
RESULTS AND CONCLUSION: (1) The human induced pluripotent stem cells expressed high-levels of the induced pluripotent stem cell-specific markers (OCT4, NANOG, and SSEA4) and self-renewal marker (Ki67) as well. (2) The human induced pluripotent stem cells were successfully induced sequentially into to cardiac mesoderm cells, endothelial progenitor cells and final typical endothelial cells. (3) Real-time quantitative PCR results showed that with the progression of the differentiation, endothelial progenitor cell markers (KDR and CD34) were increased, and then gradually decreased; and endothelial cell markers (VE-CADHERIN, ICAM1 and PECAM1) were gradually increased. Immunofluorescence at the protein level further confirmed the increasing trend of endothelial cell marker expression. (4) Tube formation assay showed that the number of endothelial cells-generated blood vessels increased with the increase of the concentration of vascular endothelial growth factor. (5) It is concluded that an experimental protocol for the directed differentiation of human induced pluripotent stem cells into endothelial cells has been successfully established, which is expected to provide a cellular basis and experimental basis for future blood vessel construction.
Key words: human induced pluripotent stem cell, endothelial cell, differentiation, signaling pathway, endothelial progenitor cell