In Vitro Differentiation From Human Bone Mesenchymal Stem Cells Into Hepatocyte-like Cells

Hepatic disease is common in clinical practice and is the severe cause harming the health of human beings. Many patients with hepatic diseases have died because of the absence of effective therapeutic method. Hepatocellular transplantation is an effective method to treat terminal stage liver function failure. However, hepatocellular transplantation is limited by deficiency of cell source, in vitro proliferation, difficult to keep original feature afterpassage and immunologic rejection. It is urgent to search for a new hepatocyte source. Human bone marrow mesenchymal stem cells(HMSCs),a type of self-proliferate and multipotent Stem cells, have been generally used in hepatic disease research because of their advantageous characteristics such as high plasticity, low immunogenicity, being easy to be isolated, and being convenient for the expression and transfection of exogenous genes. In recent years, more and more studies have shown that human bone marrow mesenchymal stem cells can differentiate into hepatocyte-like cells, under suitable microenvironment. They are being increasingly considered for future applications in liver cell therapy. It is a very good prospects that HMSCs transplantation be used in the teatment of acute and chronic liver failure. Mesenchymal stem cells transplantation is currently regarded as a possible alternative to orthotopic liver transplantation (OLT), it brings the hope for the cure of hepatic failure. So, the purpose of this experiment is mainly to study the ability of HMSCs differentiation into hepatocyte-like cells when culture in the induction system in vitro. The research could be divided into 2 parts. The first part study the biological feature of HMSCs and find a cell isolation and expansion method, so as to provide a cell source for the following studies. The second part investigate the feasibility of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) to induce the differentiation from HMSCs into hepatocyte-like cells. The expressions of hepatic markers, AFP and CK18, were detected by immunocytochemical staining and Albumin levels in culture supernatants were determined by ELISA at different times after induction. The expressions of AFP, ALB mRNA were detected by RT-PCR assay after induction. Try to find a new theory for hepatocellular transplantation and therapeutic method for liver function failure.PartⅠThe biological characteristics of human bone marrow-derived mesenchymal stem cellObjective To establish a method for isolation and cultivation of bone mesenchymal stem cells from human in vitro and explore their biological properties in order to provide an experimental basis for applying HMSCs to achieve the therapeutic benefit as seed cells in decompensated cirrhosis and liver function failure.Methods HMSCs were obtained from patients with esophageal cancer and were separated by density gradient centrifugation combined with attachment method. The configuration was observed under phase-contrast microscope consecutively and the cell growth was measured. The phenotypes of HMSCs were identified by flow cytometry.Results The subcultured cells showed active proliferative ability. More than 95% of subcultured HMSCs were adhesive in 12h. The HMSCs began to increase at 4 days. Primary culture HMSCs are proliferated in dispersion and clone settlement ways, then cells showed long spindle shape, polygon, and so on. The HMSCs did not express hematopoietic cell CD34 and CD35, but strongly expressedβ1-integrin CD29 and matrix receptor CD44.Conclusion HMSCs is different from human bone marrow hematopoietic cells. HMSCs can be cultured and purified in vitro by density gradient centrifugation and adherent culture. HMSCs showed stable and rapid growth in vitro. All the characteristics make it possible to HMSCs to be the seed cells for tissue engineering.PartⅡinduce human bone marrow mesenchymal stem cells differentiation into hepatocyte-like cells in vitroObjective To investigate the feasibility of hepatocyte growth factor(HGF) and epidemal growth factor(EGF) to induce the differentiation from HMSCs into hepatocyte-like cells in vitro. To provide seed cells for Hepatocellular transplantation and bioartificial liver.Methods HMSCs were obtained from patients with esophageal cancer and were separated by density gradient centrifugation combined with attachment method. The third-passage HMSCs were divided into four groups:HGF (adding 20μg/L HGF), EGF (adding 20μg/L EGF), HGF+EGF, and blank control groups. Morphology was observed using inverted microscope. At days 7 and 14 after induction,α-fetoprotein and albumin mRNA expressions were detected using RT-PCR assay. The expressions of hepatic markers, AFP and CK18, were detected by immunocytochemical staining and Albumin levels in culture supernatants were determined by ELISA at days 7,14,21, and 28 after induction,Results HMSCs transformed into hepatocyte-like cells gradually and changed from long fusiform shape to polygon or similar round shape in the HGF, EGF and HGF+ EGF groups. At days 7 and 14 after induction,α-fetoprotein and albumin mRNA expressions were positive. However, polygon cells were not observed in the blank control group, andα-fetoprotein and albumin mRNA expressions were negative. The expressions of AFP and CK18 were positive as detected by immunocytochemical staining. HMSCs-induced Alb production increasing in a time-dependent manner after induction in each group except blank control group and the difference was significant in the HGF,EGF and HGF+EGF groups(F=9160.446, P=0.000).Conclusion HGF, EGF, and HGF+EGF could induce the differentiation from HMSCs into hepatocyte-like cells; however, The effect of HGF+EGF is among the strongest.Through above the experimental study, we've grasped experimental skills, such as cell culture, abstraction and separation stem cell. HMSCs have the biological characteristics of extremely self-renew and multiplex differentiation potential, and also precondition of being seeding cells. After induction by hepatocyte growth factor and epidemal growth factor in vitro HMSCs not only turned into hepatic cell morphologically, but also they could express hepatocyte trait protein like AFP and ALB. So we primarily deduce that HMSCs have the ability to differentiate into hepatocytes. HMSCs used for cell transplantation, it have very important theoretical significance and clinic value.