| Umbilical cord blood(UCB)as a available source of hematopoietic stem cell transplantation(HTC)has many advantages,but the lack of hematopoietic stem cells(HSCs)have limited its clinical application.Ex vivo expansion of UCB-HSCs is a immediate and convenient way to solve the problem maintained above.HSCs first appeared in main arteries such as the vitelline and umbilical arteries.And there are still a large number of HSCs in the umbilical cord until delivery.So it is very likely underline the important factor in maintaining HSCs self-renewal.Endothelial cells(ECs)and HSCs are both originated in hemogenic endothlium(HE),it play the role of maintaining the hematopoietic homeostasis during the hematopoiesis development.Therefore,we speculate that HuAECs is one of the important microenvironmental factors affecting HSCs,and may contain elements to maintain its self-renewal and promote its proliferation.HSCs differentiated from induced pluripotent stem cells(iPSC)are another answer to the problem.Considering its epigenetic memory from their donor cells,iPSC derived from ECs which are closely related wtih HSCs may be more likely to induce to hematopoietic stem/progenitor cells(HSPCs).One of the earliest confirmed sites of definitive hematopoiesis was in the dorsal aorta within the AGM regions,and then the first adult HSCs were found in the aorta,one of which was the umbilical arteries.We conclude that the iPSC obtained by reprogramming human umbilical arterial endothelial cells(HuAECs)may retain the epigenetic memory of donor cells,and it is more likely to differentiate into HE and HSCs.To solve the problems in these areas,we conducted two studies:on the one hand,HuAECs expressing E4orfl and green fluorescent protein(GFP)stably have been established by retroviral system,as feeder cells to HSCs amplification.E4orfl can activate the Akt and FGF-2/FGF-R1 signal pathway to enable cells to survive in the serum-free environment,making ECs provide appropriate support environment for HSCs ex vivo amplification.The cytokines-alone and E4orfl/HuVECs,E4orfl/HFLSECs was used as control group for the experiment,to examine the effect of augment.On the other hand,we reprogrammed HuAECs into iPSC(iPSC-A1)with CytoTuneTM-iPSC 2.0 Sendai Reprogramming Kit.Sendai virus is RNA virus that does not integrate into host DNA,ensuring the safety of transfection.Subsequently,mesoderm,HE,and hematopoietic cells have been successive generation based on the timed addition of cytokines.As a result,the total nucleated cells(TNC)and the CD34+CD90+ HSPCs of UCB-derived CD34+ cells increased 560,240.34 folds respectively within 14 days by co-cultured with E4orf1/HuAECs.Furthermore,HSPCs still have multi-lineage differentiated capability and definitive engraftable potential in vivo,in which E4orfl/HuAECs has an advantage over the orthers.It is demonstrated that with the support of E4orfl/HuAECs,HSCs can maintain its multi-lineage and sufficient engraftment potential.At the same time,we successfully obtained iPSC-A1 by reprogramming HuAECs.iPSC-A1,which endogenous pluripotent gene raised and endothelial genes down-regulated,could maintain the normal diploid karyotype,and had been divided into triploblastic cells in the formation of the embryoid body(EBs)and teratoma.HE,which has been successive generation via stepwise differentiation cultures from iPSC-A1,could not only produce CD45+ and CD43+ hematopoietic cells but also generate the tubular structure ex vivo after differentiated into ECs.Demonstrating the potential of HE for bidirectional differentiation to hematopoietic and endothelial cells.Consequently,we established the endothlium microenvironment to co-culture with HSCs ex vivo.Meanwhile,the novel iPSC have been obtained for the production of HSCs.This provides a new and viable method for the expansion and generation of HSCs ex vivo.It is also important to have further understanding of hematopoietic ontogeny,which is significance for uncovering the mechanism of various signaling pathways in HSCs biology. |
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