Role Of Enforced Expression Tcfec In Hematopoietic Stem Cells

In hematopoietic system, transcription factor Tcfec is preferentially expressed in hematopoietic stem cells(HSCs) and multipotent progenitor cells(MPPs). Until now, the role of Tcfec in HSCs in vivo remians elusive. Object:1. Verify predominant expression of Tcfec in HSCs and MPPs.2. Establish retrovirus ectopic expression system.3. Based on enforced expression system in vivo and in vitro, analyze the effects of overexpressing Tcfec in HSCs. Methods:1. High purity HSCs and MPPs were sorted out for RNA-Seq with flow cytomer. In assistant with bioinformatics, the differential expression profile between HSCs and MPPs were obtained.2. The relative expression level of Tcfec between HSCs and MPPs were confirmed by q-PCR.3. Open reading frame(ORF) of Tcfec was established into retrovirus system. Recombinant retrovirus vector was tested via transfection and q-PCR.4. Retrovirus carried with Tcfec gene were transduced into enriched Lin- cells from E14.5 fetal liver, 40000 GFP+ nucleared cells(CD45.2) were sorted out at 48 h after infection and transplanted into lethally irradiated recipient.5. The donor reconstitution percentage and lineage composition of recipient in hematopoietic tissues were analysized post transplantation by flow cytometry.6. By means of flow cytometry, the impact of Tcfec on cell cycle of HSPC was explored.7. According to CFU aaasy in vitro, the effects on colony forming potential were assessed. Results:1. Tcfec in HSCs is relatively superior expressed than MPPs.2. The retrovirus ecotopic expression system of Tcfec was successfully build.3. Compared to the GFP control, overexpression of Tcfec in HSCs showed higher donor reconstitution,but multiple lineages contributed in Hematopoietic tissues are normal at 16 weeks after transplantation.4. Ectopic expression of Tcfec increased abosolute number of hematopoietic stem/progenitor pool, but sustained normal level in CMP, GMP and MEP.5. Cell cycle analysis of HSPC in bone marrow of Tcfec are consistent with GFP control.6. The colony number arising in GEMM from Tcfec group was more than the GFP control.7. Feeder cells AFT024 overexpressed with Tcfec co-cultured with HSCs showed no effect in engraftment. Conclusion:1. Enforced expression Tcfec enhance engraftment of Hematopoietic Stem Cell.2. HSCs overexpressing Tcfec maintain self-renewal and multipotent differentiation ability.3. Tcfec is a key functional molecule in regulating engraftment of hematopoietic stem cells.