The Investigations Of Multipotent Adult Progenitor Cells From Human Bone Marrow Differentiating Into Hepatocyte-like Cells In Vitro

The treatment of liver diseases, such as hepatitis, liver cirrhosis and liver cancer, is always a global difficulty. To end-stage liver failture led by these disorders, orthotopic liver transplantation(OLT) is the only established treatment, but it is associated with donor shortage, immune rejection, and high cost, which are the limitations of this therapy. The treatment purpose of Hybrid bioartificial liver(HBAL) to the end-stage liver failure patient is bridged for OLT. On the other hand, the function of hepatocyte repopulation is mighty, applied HBAL temporarily may obtain some purpose of treating anxious liver function, accelerating hepatocyte repopulation, supporting liver function renenw, and healing the state of an illness. However, the anticipated clinical efficacy of HBAL has not proved yet, mainly because of the source deficiency of hepatocytes which are the core bioartificial materials of HBAL.The investigation have proved that multipotent adult progenitor cells(MAPCs) from human bone marrow can differentiate into functional hepatocyte-like cells. MAPCs may be a relative ideal cells for liver tissue engineering as they are more easily to be manipulated, have the merits of abundant donor source and easy to harvest, and be obtainted autologously to avoid immune rejection. Part I : The investigations on purification and culture of multipotentadult progenitor cells from human bone marrow Objective To create a method of separating human MAPCs in vitro using immunomagnetic beads. To investigate the possibilities of MAPCs-5-producting and proliferating by cell culture produced by ourselves.Methods The bone marrow mononuclear cells were obtained by inspiring the proper volume of bone marrow from volunteers and then centrifuging by gradient and density. The mononuclear cells were cultivated by adherent culture. Collected the bone marrow adherent cells, and isolated the adherent cells by MACS through depletion selection by use of CD45 and GlyA microbeads. Analyzing the component of the CD45~>. GlyA" cells by flow cytometers. Observed the cells' form hi different growing phase. Flow cytometers analyzed the subculture cells at different time point in order to investigate the immunization stability of MAPCs producting and proliferating by cell culture.Results (1) Per lX106/ml bone marrow mononuclear cells could separated 5 ?10X104/ml MAPCs by MACS. The activity of the pro-purification or after purification cells was (96.7?.7) %and (96.0?.4) % respectively, which was not different significantly with that before purification. (2) The MAPCs grew well in the cell cultures produced by ourselves. We observed that MAPCs could be expanded for more than 16 population doubling. (3) The purity of the CD45~% GlyA~cells separated from bone marrow adherent cells was more than 98 % by flow cytometers. The purity of MAPCs which had cultivated 4, 8 and 12 cell doublings were more than 98 % by flow cytometers.Conclusions (1) The MAPCs derived from bone marrow can be purified by MACS through depletion selection by use of CD45 and GlyA microbeads, and they can survive and differentiate after tune. (2) The abilities of MAPCs producting and proliferating by cell cultures is active, and can keep non-differentiation state for a long time. (3) MAPCs cultures produced by ourselves is appropriate MAPCs culture in vitro.Part II: The investigations of multipotent adult progenitor cells fromhuman bone marrow differentiating into hepatocyte-like cells in thedifferent appropriate condition in vitroObjective To investigate the possibility of the MAPCs from human-6-bone marrow which are induced into hepatocytes in vitro. To explore the feasibility of differentiation from the MAPCs into the hepatocytes, and to establish the method and condition of MAPCs culture in vitro. And then, to make the hepatocytes derived from MAPCs as germ cell to be useable in tissue-engineering and clinical medical.Methods (l)Divided groupsrA: Hepatocyte growth factor (HGF, 20ng/ml), B: Fibroblast growth factor-4(FGF-4 , 20ng/ml),D: HGF(20n...