Study On The Role Of Sall4 In Somatic Cell Reprogramming And Hoxd12 In The Pluripotency Of ESCs

Induced pluripotent stem cells(iPSCs)can differentiate into all somatic cell types in vitro,leading to iPSCs are often expected to be used in the repair of organ damage and the regeneration of necrotic tissue.However,the problems such as low induction efficiency,long induction cycle,and clonal heterogeneity caused by insufficient understanding of the reprogramming mechanism have become the main obstacles restricting the clinical application of iPSCs.Therefore,explaining the reprogramming mechanism of somatic cells has become an urgent problem for scientific researchers.Previously,our laboratory developed a group of high-efficiency reprogramming systems that uses 7 transcription factors(Sall4-Glis1-Mkk6-NanogKdm2b-Jdp2-Esrrb,7 factors)to induce pluripotency,and found that Sall4 plays an indispensability role in this system.However,the mechanism of how Sall4 mediates cell reprogramming in 7 factors is largely unknown.Therefore,this study used7-factor induced somatic cell reprogramming as a model,combined with bioinformatics analysis,gene overexpression,gene knockdown technology,and explored the mechanism of Sall4 mediated 7-factor reprogramming.The results showed that: in the process of 7 factors inducing pluripotency,Sall4 activated the expression of limb development-related genes Hoxd11,Hoxd12 and Hoxd13.Further studies found that knockdown of Hoxd12 significantly reduced the efficiency of 7factors induced somatic cell reprogramming,but knockdown of Hoxd12 had no significant effect on the somatic cell reprogramming induced by Yamanaka factor(Oct4-Klf4-Sox2).In addition,at different stages of reprogramming induced by 7factors and Yamanaka factor,overexpression of Hoxd12 respectively had no significant effect on somatic cell reprogramming;substitution experiments proved that Hoxd12 could not replace or partially replace the function of Sall4 mediated cell reprogramming.To study the function of Hoxd12 in mouse embryonic stem cells(mESCs),this study used CRISPR/Cas9 gene-editing technology to construct a mouse embryonic stem cell line with knocked out of Hoxd12,and proved that knocking out Hoxd12 does not affect the expression of pluripotency genes in mESCs,but knocking out Hoxd12 can delay the proliferation rate of mESCs.This study initially explored the mechanism of Sall4 mediated 7-factor reprogramming,and found that Hoxd12 plays an important role in regulating somatic cell reprogramming and the proliferation of mESCs,providing a reference for revealing the mechanism of Sall4 mediating cell reprogramming,and promoting the safety clinical application of iPSCs.