ABSTRACT
Objective To generate hemogenic endothelial cells(HECs)from human induced pluripotent stem cells (hiPSCs)in vitro in order to learn more about the mechanism by which the vascular niche affects HECs production and self -renewal.Methods hiPSCs with reporter gene runx1c were differentiated to hematopoietic cells by spinEB method.The CD34 positive cells were sorted by magnetic-activated cell sorting(MACS)at day 10 after hematopoietic differentiation. Afterwards,these CD34 positive cells were co-cultured with DLL4 overexpressed vascular niche cells VeraVec to further differentiate to HECs.The HECs derived from the hiPSCs were characterized by FACS.Results We first established an hiPSCs single cell culture method for spinEB differentiation.Single cell cultured hiPSCs with reporter gene runx 1c were differentiated to form embryonic bodies(EBs)by spinEB method.The HECs were enriched from the day 10.Meanwhile, we cultured the E4ORF1 transfected human umbilical vein endothelial cell(HUVEC)line(VeraVec)and examined the expression of NOTCH signaling pathway related genes.According to the results, VeraVec had a high expression level of NOTCH ligand DLL4 at both mRNA and protein levels.And the CD34 positive HECs were co-cultured with DLL4 overexpressed VeraVec cells,which promoted the expression of tdTomato during hematopoitic differentiation and increased HSCs production.Conclusion A method of inducing hiPSCs differentiation by spinEB has been established, which can enrich HECs.This model can be applied to study the mechanism by which the vascular niche promotes hematopoietic differentiation from hPSCs.The generated functional HSCs are of great social and military values for HSCs transplantation and battlefield radiation injury treatment.