Culture And Difierentiation Liver Progenitor Cells Toward Hepatocytes In Vitro

Backgroud:At present, orthotopic liver transplantation is the only curative treatment for terminal liver diseases. However it is limited by a significant shortage in donor livers. Bio-artificial liver support systems, as an alternative therapy, have been developed to bridge unstable patients until a liver transplant is available, or to allow patients to recover from acute liver failure, but require an enormous number (above5×109) of functional liver cells. Hepatocytes are most often used since they are the major parenchymal cells in the liver. Isolated hepatocytes are mainly obtained from cadaveric donor livers, but these are only available in limited numbers. In addition, it is difficult to expand hepatocytes and to maintain their correct physiological functions in vitro. Hence, other cell types that can be extensively expanded ex vivo, such as embryonic, adult or induced stem cells, and liver progenitor cells (LPCs), have attracted much attention as potentially superior alternatives to hepatocytes.Liver progenitor cells, identified as small ovoid cells with high nuclear to cytoplasmic ratio, are rare in normal livers, but expand transiently from the terminal bile duct system, differentiate into hepatocytes and biliary cells and infiltrate into the liver plates to play an important role in liver regeneration when the replication of mature liver cells is significantly impaired. As the resident progenitor cell in the liver, LPCs are developmentally closer to the hepatocyte and cholangiocyte than embryonic or bone marrow stem cells and would be able to differentiate more rapidly into functional cells that are urgently required. This reason plus their capacity for extensive replication in vitro and their bi-potentiality, means LPCs are worthy of serious consideration in cell therapy applications such as liver cell transplation, liver tissue engineering, gene therapy and bio-artificial liver support for a host of liver diseases. However, several issues have to be addressed to keep the promise of cell therapy. To establish safe and efficient ex vivo culture systems for the expansion and differentiation of LPCs is clearly essential.Part I Optimization of serum-free culture conditions for the expansion and differentiation of liver progenitor cells using different collagen proteinsObjective:To optimize serum-free culture conditions for the expansion and differentiation of LPCs using different collagen proteins. To evaluate how various types of collagen affect on the morphology, attachment, proliferation and differentiation of LPCs.Methods:Bipotential mouse embryonic liver cells from a TAT promoter-lacZ transgenic mouse (BMEL-TATs) were used as a model to optimize ex vivo serum-free culture condition for the expansion and differentiation of LPCs. Expression of this reporter gene correlates with the differentiation and maturation of the LPC line with regard to the hepatocyte lineage. The morphology, attachment, proliferation and differentiation of BMEL-TATs were evaluated on tissue culture plates (TCPs) coated with different collagens; namely I, III, IV as well as the mixture of collagen I and III (1:1) in serum-free medium. The BMEL-TATs cultured on the normal TCPs in the same medium, and in medium supplemented with5%fetal bovine serum (FBS) represent negative and positive controls respectively. Cell morphology was observed by light microscope and the cell attachment was calculated by attached cell to total seeding cell ratios at different time points. Cell proliferation was measured with a Cellscreen system based on the area occupied by the cells, as well as haemocytometer counts. The semiquantitative staining and quantitative fluorimetric measurement of β-galactosidase activity were used to evaluate differentiation of LPCs towards hepatocytes.Results:LPCs grew as a monolayer on all the tested collagens, but became aggregates on TCPs without collagens in the serum-free medium. Various types of collagens promoted their attachment, proliferation and affected differentiation equally, the effects of which were comparable to adding5%FBS to the medium. Interestingly, the cells grown as aggregates showed higher differentiation than those growing in a monolayer, indicating cell aggregates with3-dimensional structure best facilitated LPC differentiation without the need for a substrate of extracellular matrix (ECM).Conclusions:All tested types of collagen-coated TCPs enabled BMEL-TATs to survive and expand in a serum-free medium. Additionally, a three-dimensional culture method, not requiring ECM components may be the best approach to differentiate LPCs into functional hepatocytes. Part Ⅱ Effects of alginate-chitosan microencapsulation on the ex vivo differentiation of liver progenitor cells towards hepatocytesObjective:To evaluate the feasibility and effectiveness of alginate-chitosan (AC) microencapsulation for the ex vivo differentiation of LPCs towards hepatocytes.Methods:BMEL-TATs were used as a model to detect the effects of AC microencapsulation on the ex vivo expansion and differentiation of LPCs towards hepatocyte lineage. Two different initial cell densities,5×106/ml and2×106/ml, were tested in the study. We cultured AC microencapsulated BMEL-TATs either in the growth medium for21days or in the differentiation medium for15days after three days'culture in the growth medium. The cell morphology, viability, proliferation and differentiation, and the mechanical stability of AC microencapsules were evaluated dynamically. The cell morphology was observed by light microscope and laser confocal microscope. The viability of LPCs was determined by FDA-PI double staining. Cell proliferation was measured with haemocytometer. The detection of hepatocyte, cholangiocyte and LPC mark gene expression in the cells by real-time PCR, and the measurement of urea in the culture medium were used to evaluate differentiation of LPCs towards hepatocytes. The BMEL-TATs cultured on normal TCPs served as control.Results:The mechanical stability of AC microcapsules with BMEL-TATs to withstand the shear stress induced by high agitation rate was well presented. BMEL-TATs showed high viability in the AC microencapsules. Increased number and size of BMEL-TAT aggregates were found in the microencapsules during the process of culture or differentiation. The proliferation of BMEL-TATs in the microcapsules was limited, whereas their differentiation towards hepatocytes was better than those cultured on normal TCPs. The microencapsuled BMEL-TATs present better differentiation when encapsuled with high cell density (5×106/ml), rather than with low cell density (2×106/ml).Conclusion:AC microencapsulation can promot the differentiation of BMEL-TATs towards hepatocytes in vitro. It is better to choose5×106/ml as encapsuled cell density to achieve higher differentiation.