| Mesenchymal stem cells(MSCs)were first found and identified in bone marrow,and then successively identified in a variety of tissues of human embryos and adults,including adult bone marrow,fat,synovial membrane,bone,muscle,lung,liver,pancreas,amniotic fluid,umbilical cord and umbilical cord blood,etc.Studies have shown that although mesenchymal stem cells have different tissue sources,their clinical application prospects have attracted more attention due to their common stem cell characteristics and immune regulatory capacity.At present,bone marrow-derived mesenchymal stem cells are more studied in clinical application,but its preparation process is not easy to carry out quality control,and it is harmful to patients when taking materials.It is not suitable for allogeneic transplantation due to its strong immunogenicity;In addition,the number of cells,their proliferative capacity and differentiation potential are negatively correlated with the age of the donor,which limits the clinical application of bone marrow mesenchymal stem cells.By contrast,mesenchymal stem cells isolated from placental and umbilical cord tissues not only maintain the biological characteristics of mesenchymal stem cells,but also have the characteristics of large differentiation potential,strong proliferation capacity,low immunogenicity,convenient sampling,no limitation of moral and ethical problems,easy industrial preparation and the like,and thus are likely to become pluripotent stem cells with more clinical application prospects.The liver is the most critical metabolic center in the human body,and drugs,viruses,and toxins often cause liver damage or even liver failure.Cell therapy can partially solve the problem of insufficient source of liver donors.At present,the functional hepatocytes under research come from various sources,such as obtaining primary hepatocytes from liver tissue,cell reprogramming,and inducing differentiation of pluripotent stem cells or tissue stem cells.However,these methods also have the problems of complex technology,long cycle,and high heterogeneity of cell products.Human Wharton’s jelly-derived mesenchymal stem cells(h WJ-MSCs)are mesenchymal stem cells isolated from human umbilical cord Wharton’s jelly,which are the most abundant and attractive source of mesenchymal stem cells and can be obtained by non-invasive methods.At present,a variety of chemical induction of MSCs into cells of ectodermal,mesodermal and endodermal lineage has been reported,which shows the multi-directional differentiation potential of MSCs as stem cells.To date,there have been many research reports on the scheme of inducing human umbilical cord mesenchymal stem cells(h UC-MSCs)to differentiate into hepatocytes,but there is still great room for optimization in terms of applicability and induction efficiency.In particular,the repeatability of many induction schemes needs to be further evaluated.However,these research results together prove that the induction scheme using small molecular compounds with clear action targets is a method with clinical application prospect.Sodium butyrate is a small molecular short-chain sodium salt of butyric acid.It is essentially a Histone deacetylase inhibitors(HDACi),which can be used to inhibit tumorigenesis,treat nervous system diseases,and induce stem cell differentiation under physiological conditions.The Wnt/β-catenin signaling pathway widely exists in the evolution of multicellular organisms,with a high degree of conservation and cell dependence,and plays an important role in regulating the proliferation and growth of cells.Small molecule inhibitors of glycogen synthase kinase-3β(GSK-3β)can protect the substrate β-catenin of GSK-3β from degradation.β-catenin accumulates in a large amount in the cells and transfers to the nucleus,which then initiates the expression of target genes and promotes the differentiation of stem cells.CHIR99021 is a small molecule inhibitor that can be used as an agonist of Wnt signaling pathway and maintain the active state of Wnt pathway by inhibiting the activity of GSK-3β.Multiple studies have shown that CHIR99021 promotes self-renewal of mouse and human Embryonic stem cell(ESCs)and also plays an important role in reprogramming.In this study,the combination of small molecular compounds was used to induce h WJ-MSCs into hepatocyte-like cells.The main work as follows: 1)First,through primary culture,a fibroblast-like cell line was isolated and established from human umbilical cord Wharton’s jelly.Through identification,it was confirmed that the surface protein expression of the obtained fibroblast-like cell line met the basic expression characteristics of MSCs and had the potential of osteogenic,adipogenic and chondrogenic differentiation,so it was defined as h WJ-MSCs.2)The gene expression was detected by RT-PCR,and the results showed that h WJ-MSCs not only expressed interstitial cell gene and epithelial cell gene,but also expressed early hepatic genes GATA4,GATA6,HNF4 a and Ep CAM in embryonic development,but did not express mature hepatocyte genes ALB,CYP3A4,CYP1A1 and other functional genes.3)Three hepatic induction schemes of MSCs reported in the literature were verified,and the results showed that these schemes had poor induction effect and were not suitable for hepatic induction of our strain of h WJ-MSCs.4)The ability of some growth factors to initiate MSCs hepatic transdifferentiation was identified,and it was found that the combination of Epidermal growth factor(EGF)and Basic fibroblast growth factor(FGF2)could promote the expression of Albumin(Albumin,ALB)in MSCs cells.In combination with the literature,a new induction scheme for MSCs hepatic transdifferentiation was established,and the combination and concentration of growth factors and small molecular compounds were optimized.The cells were pretreated with a combination of Sodium butyrate(Na Bu),EGF,and FGF2,and then differentiated into hepatic precursor cells using CHIR99021 in combination with EGF and FGF2,as well as Hepatocyte growth factor(HGF),Insulin-transferrin-selenium-ethanolamine(ITS-X)and FGF2.Finally,oncostatin M(OSM),Dexamethasone(Dex)and ITS-X were used to induce further differentiation into mature hepatocytes.Through identification,we found that after induction,the cells underwent significant morphological changes,transforming from original fibroblast-like cells to epithelioid cells,with typical dual nuclei of hepatocytes.Gene expression tests at different induction time points showed that liver precursor cell markers,including AFP and HNF4a;were expressed in the intermediate stage of induction.Later,mature hepatocyte markers including ALB and CYP3A4;were gradually expressed.And also expresses an epithelial cell marker E-Cadherin.5)The influencing factors of hepatic induction were preliminarily explored,and it was found that Na Bu and CHIR99021 exhibited concentration-and time-dependent effects in hepatic induction.In conclusion,a new hepatic induction protocol of h WJ-MSCs was established in this study,which laid a foundation for further optimization and in-depth study on the differentiation mechanism. |
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