Epigenetic Regulation Mechanism In The Induction Of Transdifferentiation Of T Lymphocytes To B Lymphocytes

Cell differentiation has been considered a one-way irreversible process for a long time.With the development of life science research,researchers have successfully reversed differentiated adult cells into pluripotent or totipotent cells by nuclear transfer,cell fusion,and the introduction of specific transcription factors,enabled reprogramming of somatic cells.In 2006,the birth of induced pluripotent stem cells(iPSC)pushed the progress of somatic cell reprogramming to its peak.Researchers at the beginning derived iPSC from mouse fibroblasts,subsequently from human fibroblasts,and then from neural progenitor cells,skin cells,spermatogonia,hair keratinocytes,pancreatic?cells,and lymphocytes.They obtained iPSC first with four transcription factors(Oct4,Sox2,Klf4,and c-Myc,OSKM),afterwards with only two or three transcription factors,then with transcription factors combined with small molecule compounds,and finally with even only a combination of small molecule compounds,the iPSC obtained by the last way called chemically induced pluripotent stem cells(CiPSC).Small molecules can not only improve the efficiency of reprogramming,but also specifically regulate certain proteins because of their relatively clear target,easy operation,and strong controllability and specificity and some of them can partially or completely replace the transcription factor,so more and more stem cell researchers have began to focus on study with small molecules.The technology of iPSC provides the opportunity to form cells that resemble embryonic stem cell(ES)in its properties,like pluripotent and potentially unlimited self-renewal.Studies have shown that iPS cells can differentiate targetedly into various types of functional cells in vitro and the therapeutic effects of transplanted iPS cells have been demonstrated in a variety of animal models.With the development of science and technology,iPS technology will definitely bring broad prospects for cell therapy,drug screening and disease mechanism research.Cell transdifferentiation refers to the transformation of a mature,terminally differentiated cell into a different one by transfection of key transcription factors which are necessary for the development of target cells.Many studies have reported the results of related work on transdifferentiation,such as the transdifferentiation of human fibroblasts into dopaminergic neurons,mouse B lymphocytes into macrophages,and terminally differentiated mouse hepatocytes into neurons.Epigenetic regulation plays an important role in reprogramming.Therefore,small molecule compounds that regulate epigenetic have a great impact on reprogramming.Histone deacetylase(HDAC)inhibitors and DNA methyltransferase inhibitors perform the epigenetic regulation by affectting the function of histone deacetylase and DNA methyltransferase.The main purpose of this study is to explore the role of epigenetic modification during the transdifferentiation of T lymphocytes.In this study,three epigenetic modulators,histone deacetylase inhibitors(VPA,TSA)and a methyltransferase inhibitor(5-aza-2'-deoxycytidine)—were used to study the changes of T lymphocyte(Jurkat)under the effect of the inhibitors,and the changes of cell phenotype were detected at the mRNA and protein levels by real-time quantitative PCR and flow cytometry respectively.The results showed that after the treatment by the three small molecular epigenetic inhibitors,Jurkat cells begin to express B cell phenotype,and the stem cell marker also began to express.According to the level of expression of each detected index,we determined the optimal combination of small molecular compounds—VPA and 5-aza-2'-deoxycytidine(V5)—for the next step for the research.We further optimized the induction method,OP9 cells(murine bone marrow stromal cells which have a genetic defect in macrophage colony stimulating factor can support the growth of B cells by restricting myeloid development)were added afer the treatment of V5,the changes of cells in different levels were detected by real-time quantitative PCR,flow cytometry,ELISA,Western blot,CFSE and,high-throughput sequencing.Under the optimized conditions,the transdifferentiation of Jurkat cells into B lymphocytes was found to be more obvious and the characteristics of stem cells were also more apparent.Then,we briefly explored the molecular mechanism of transdifferentiation of T lymphocytes into B lymphocytes.BSP sequencing and Ch IP were used respectively to detect the changes of DNA methylation and histone acetylation in the promoter region of PAX5 which is a transcription factor regulating B cell development and maintaining B cell identity.The results showed that histone deacetylase inhibitors regulate the expression of B lymphocyte transcription factor PAX5 by up-regulating the acetylated H3K9,thereby promoting the development of B cells.DNA methyltransferase inhibitors promote the transdifferentiation of Jurkat cells into B lymphocytes by inhibiting the methylation level of CpG islands in the PAX5 promoter region.We performed the same treatment in primary CD4~+T cells and obtained consistent results with those got in the research with Jurkat.These results suggest that epigenetic inhibitor can regulate T lymphocyte transdifferentiate into B lymphocyte.Our study add a powerful evidence on the transdifferention of human cells and provids an important reference for the clinical application of epigenetic modification enzyme inhibitors in antitumor therapy.