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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.
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Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.
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Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.