Generation Of Human Hepatocyte-like Cells From Hair Follicle Mesenchymal Stem Cells–derived IPSCs Through Definitive Endoderm

BackgroundAs well-known, i PS cells are similar to ES cells, such as morphology, expression of specific genes, and have the potentiality of self-renewal and multi-directional differentiation. It avoided the ethical issues faced by ES cells. The successful cases have been reported in the literature about generation of hepatocyte-like cells from i PS cells. But there are still many problems have to be solved.First of all, the selection of target cells is critical. Recently, there are many sources of i PS cells, such as, skin fibroblasts, mouse embryonic fibroblasts, adipose stem cells, umbilical cord blood derived-CD34+ cells, umbilical cord mesenchymal stem cells and so forth. We chose human follicle mesenchymal stem cells as the source of human induced pluripotent stem cells. It has several advantages, such as extensive materials, safer and more noninvasive.Secondly, the traditional induction method is inducing i PS cells to hepatocyte-like cells through the embryonic stage(EB). This is aim at simulating the process of embryonic development in vivo. However, some studies have confirmed that the three-dimensional structure makes the differentiation of cells containing a large number of cells in different stages of differentiation. It goes against the principle of efficiency and reduces the efficiency of induction. Therefore, we must explore a new method of induction. We induced human hair follicle mesenchymal stem cells–derived i PS cells to hepatocyte-like cells through definitive endoderm intermediate. This method improves the effi ciency of the induction, the life of cells and the number of cells.Finally, the existing methods of induction still have low efficiency. Therefore, we have improved the method of induction. We added a large dose of HGF, so as to effectively improve the efficiency of definitive endoderm.MethodWe explored the approach of generating patient specific human hepatocytes from human induced pluripotent stem cells(i PSCs) through definitive endoderm.(a) We reprogrammed human hair follicle mesenchymal stem cells to construct hair follicle derived-i PS cells. It is safer and noninvasive.(b) Compared with the traditional induction method, our method which is through definitive endoderm improves the purity and longevity of the cells, and greatly improves the efficiency of the induction.(c) We added a large dose of HGF, so as to effectively improve the efficiency of definitive endoderm.ResultsWe used the phase contrast microscopy to observe human hair follicle derived-i PS cells. The results of RT-PCR confirmed that it does not express the specific marker genes of DE and HLCs. we can observed that breakage of cell-cell junction in DE. It also expressed the specific marker genes of DE, such as SOX17 and FOXa2. In the light microscope, HLCs were polygonal and round nucleus. It expressed the specific marker genes of HLCs, such as ALB,AFP,HNF4α,AAT,CYP3A4 and CK18. Immunofluorescence staining showed that ALB, AFP, CYP3A4 and CYP7A1 were expressed in HLCs. HLCs also have the function of glycogen storage, LDL uptake and ICG excretion.ConclusionWe chose human follicle mesenchymal stem cells as the source of human induced pluripotent stem cells. It is safer than the others. We induced i PSCs to hepatocyte-like cells through definitive endoderm to improve the efficiency of induction. We added large doses of HGF to improve the efficiency of generation of definitive endoderm.We can produce a large number of i PS cells derived-hepatocytes, and bring the hopes of life to patients who have liver failures.