Cytologic Analysis And Differentiation Into Hepatocyte-like Cells Of ZHJ-MAPCs

Although for end-stage liver failure derived from hepatitis, liver cirrhosis and liver cancer, etc, orthotopic liver transplantation(OLT) is most effective, it is limited by increased shortage of donor organs, immune rejections, and high cost. The experiential hepatocytes transplantation (HCT) obtained in the past 30 years has been applied in clinical trials. Initial results have shown that HCT is effective for treating chronic or acute liver failure and inborn error of metabolism of liver diease. But it is also problemed by hepatocyte shortage, immune rejection, and so on. Therefore hybrid bioartificial liver(HBAL) therapy may served as a bridge between end-stage liver failure and OLT or liver regeneration. Even though some HBAL systems proved to prolong animal survival generally, clinical results are far from expected yet, mainly due to the lack of enough hepatocyte mass. It has been demonstrated that human bone marrow derived multipotent adult progenitor cells(MAPCs) can differentiate into functional hepatocyte-like cells. MAPCs may be ideal cells for tissue engineering owing to their easy harvest and manipulation, as well as abundant donor source. Autogeneic MAPCs avoids immune rejection. In our earlier experiments, MAPCs, the subset of mesenchymal stem cells(MSCs) were successfully purified and enriched by sequential density-gradient centrifugation, selection by adherent culture and magnetic activated cell sorting(MACS). We named the cells as ZHJ-MAPCs, and established in vitro cultural methods to propagate the cells.Part â…  : Phenotypes and genetics of ZHJ-MAPCsObjective To understand the specific phenotypes and genetics on continuously cultured ZHJ-MAPCs purified by CD45 and GlyA negative magnetic activated cellsorting.Methods Continuously cultured cryopreserved original generation of ZHJ-MAPCs were analyzed for expression of CD29, CD4^ CD34 and HLA-DR, and observed for karyotypes at the 6th and 13th.Results (1)CD29 positive ratio in ZHJ-MAPCs was 99.2%, with CD44 positive ratio 98.3%, CD34 positive ratio 1.2% and HLA-DR positive ratio 5.3%. (2) Continuously cultured ZHJ-MAPCs did not show normal diploids. The number of chromosome were 55~58, accounting for 80% of all chromosomes. Chromosomal shapes were also diversified.Conclusions (l)ZHJ-MAPCs are highly purified cell with the phenotype characteristics of MSCs. (2)After continuous culture, ZHJ-MAPCs do not have normal diploids, and chromosomes have diversified shapes.Part II: ZHJ-MAPCs differentiate into hepatocyte-like cells induced byco-culture with human hepatocyte line L02Objective To investigate the possibility of ZHJ-MAPCs to differentiate into hepatocytes by co-culture with human hepatocyte line L02 in vitro. To evaluate the potential use of ZHJ-MAPCs in tissue-engineering either experimentally or clinically.Methods (1) Co-culture without cell to cell contact: ZHJ-MAPCs and L02 hepatocytes were speared on coverslip separately (both with cell density of lxlO5/ml), then they were put in a culture dish(lOcm). The expressions of ALB> AFP> CK-18> CK-19 in ZHJ-MAPCs were detected by immunocytochemistry in different time points. Seperated culture of L02 hepatocytes served as Positive control and separated culture of ZHJ-MAPCs served as negative control. (2) Co-culture with cell to cell contact: ZHJ-MAPCs labelled with CFSE were mixed with L02 hepatocytes(both with cell density of lxlO4/ml), and then the mixed cells were seeded on spearal dish for detection by Laser Scanning Confocal Microscope(LSCM). 5 days later, the cells were double stained with SABC-Cy3. The expression of ALBx AJ^ CK-18 in ZHJ-MAPCs were observed under LSCM. Similarly, Seperately cultured L02 hepatocytes served as Positive control and separately cultured ZHJ-MAPCs served asnegative control.Results (1) Results of co-culture without cell to cell contact: At the first day of co-culture without cell to cell contact, the ZHJ-MAPCs expressed high AFP. Then AFP expression tapered daily and there was hardly any expression of AFP at day 7. The expression of ALB was very weak at day 1, but increased a lot day 3, reached its peak at day 5, still maintained high level at day 7. The initial expression of CK18 appeared at day 5 and reached higher level at day 7. The expression of CK19 was always negative. The positive control cells have high expression of ALB and CK18, while weak expression of AFP and negative expression of CK19. The nagative control cells have no expressions for the four markers. (2) Results of co-culture with cell to cell contact: At the fifth day of co-culture with cell to cell contact, there were three colours of fluorescence, under LSCM: yellow Cells are ZHJ-MAPCs differentiating into hepatocytes; green Cells are undifferentiated ZHJ-MAPCs; red Cells are L02 hepatocytes. The result showed that ALB and CK18 were expressed in many cells, AFP appeared in only a few cells.Conclusions ZHJ-MAPCs can be induced to differentiate into mature hepatocyte-like cells by co-culture, either with or without cell to cell contact, but the former may be more effective.