Regulation Of Stem Cells Differentiation By Transcription Factor TEL/ETV6

The ability to generate hematopoietic stem cells(HSCs)from human pluripotent cells(hPSCs)would enable many biomedical applications.In vitro differentiation of human pluripotent stem cells to hematopoietic stem cells is a long-sought goal that would benefit patients requiring myeloablative therapy who lack an optimal matched HSC donor,and would also provide a source of lymphoid and myeloid cells for research and cell therapy.The emergence of iPSC technology can avoid the legal,ethical and moral issues involved in ESC application.More importantly,patient specific iPSC derived HSC have no immune rejection after transplantation,opened up a new direction for the implementation of individualized regenerative medicine.It is still a big challenge to induce human pluripotent stem cells differentiation into transplantable hematopoietic stem cells and mature blood cells in vitro.In this study,we started from mesenchymal stem cells(MSCs),an important member in the bone marrow microenvironment and try to identify additional transcription factors that stimulate osteogenic differentiation form hMSCs in a systematic and un-biased manner.At the same time,we clarified the role of ETV6 in osteoblast differentiation from bone marrow MSC and hematopoietic differentiation from human pluripotent stem cells in vitro.Chapter 1:The role of TEL/ETV6 in the osteogenic and adipogenic differentiation from multi-potent human bone marrow mesenchymal stem/stromal cellsAlthough several transcription factors have been identified as master regulators of osteogenic differentiation from multi-potent human mesenchymal stem/stromal cells(hMSCs),little is known about the roles of many other transcription factors that are likely also important for the osteogenic regulatory network.In the current paper,we report a novel approach to identify additional transcription factors that stimulate osteogenic differentiation form hMSCs in a systematic and un-biased manner.Our analysis revealed that overexpression of ETV6,ETV2 and LM02 promoted osteogenic differentiation of hMSCs whereas ETV3,GATA1,and HOXB4 had inhibitive effect.We focused on the effects of ETV6 expression that had the greatest enhancement on osteogenic differentiation while reduced adipogenic differentiation of hMSCs.Mechanically,ectopic expression of ETV6 in hMSCs strengthened osteogenic differentiation through upregulation of known osteogenic genes,whereas knockdown of ETV6 had the reverse effects.At last,this study also found that overexpression of ETV6 on bone marrow mesenchymal stem cells has a greater supportive function in hematopoietic stem cells expansion.Taken together,this study reveals an unexpected role of ETV6 in osteogenic differentiation and adds to a new player in the control of hMSC differentiation.Our data revealed and confirmed novel roles of ETV6 in osteogenic differentiation,in addition to the previously published discovery that it(also called TEL)is important in hematopoiesis and vascular cell formation.We believe that the new discovery will help various stem cell research by many scientists.Chapter 2:TEL/ETV6 regulates differentiation of pluripotent stem cells into hematopoietic cells in vitroThe molecular determinants regulating the specification of human pluripotent stem cells into hematopoietic cells remain elusive.TEL/ETV6 plays a relevant role in leukemogenesis and hematopoiesis.It is highly expressed in hematopoietic stem and progenitor cells(HSPCs)and is downregulated upon differentiation.Etv6-deficient mice display impaired hematopoietic development,and deregulation of ETV6 translocation and gene mutation is frequently associated with acute leukemia.In this chapter,we first established a stable and efficient in vitro differentiation system from human pluripotent stem cells into hematopoietic cells.By using the CRISPR/Cas9 technology,we knocked out ETV6 on human induced pluripotent stem cells(iPSCs),then to investigate the function of ETV6 in hematopoietic differentiation.Our data showed that ETV6 is not essential for human iPSCs maintain.ETV6 knockout did not affect the generation of hematopoietic stem and progenitor cells(HSPCs),whereas hindered the megakaryocyte differentiation from CD34+CD45+HSPC.Futhermore,we cocultured human iPSC and ETV6 overexpressed MSC,then performed hematopoietic differentiation,which promoted hematopoietic commitment of human pluripotent stem cells.It is the first study to investigate the role of ETV6 in hematopoietic cells differentiation from human pluripotent stem cells.These findings provide strong evidence for definitive HSCs generation in vitro.