The Microencapsulated Hepatocytes Prepared The Developmental Regulation Of Embryonic Stem Cell Research,

Orthotopic liver transplantation (OLT) remains the most successful treatment for many cases of end-stage liver disease. However, the efficiency of liver transplantation is limited by the shortage of available donor organs, risk of rejection, infections, and other complications caused by the lifelong immunosuppression. Liver cell therapies, including hepatocyte transplantation and bioartificial liver (BAL), are considered promising new approaches to treatment of patients with end-stage liver disease. In this project, I carried out series of studies of stem cell technology-based liver regenerative medicine:we establishment development of microencapsulated hepatocytes for bioartificial liver or transplantation in vivo directly, and investigation the molecular mechanisms regulating hES-derived definitive endoderm differentiation by miRNA-Array and proteome. Those works will offer a foundation to provide a sufficient number of seed cells for microencapsulated hepatocytes.Our study mainly includes two parts.1. Treatment of acute hepatic failure in the mouse by transplantation of mixed microencapsulation of rat hepatocytes and transgenic human fetal liver stromal cellsWe investigated the effects of transplantation of microcapsules containing a mixture of rat hepatocytes and human fetal liver stromal cells which are engineered to produce basic fibroblast growth factor (bFGF), on acute liver failure in mice. We found that different combinations of microencapsulated rat's hepatocytes and stromal cells survive, grow and function better in 3-dimensional (3-D) conditions in vitro. The metabolic activity of rat hepatocytes co-microencapsulated with human fetal liver stromal cells, particularly when engineered to produce bFGF (FLSCs/bFGF), is significantly higher than that of microcapsules with rat hepatocytes alone. In subsequent experiments, we found that the transplantation of encapsulated hepatocytes alone and with FLSCs or FLSCs/bFGF can improve the survival rate in an acute liver failure mouse model. Moreover, dramatic liver regeneration was observed after transplantation in the group that received intraperitoneal transplantations of encapsulated hepatocytes with FLSCs/bFGF. Therefore, transplantation of encapsulated hepatocytes and human FLSCs/bFGF may be a promising strategy to treat acute liver failure or related liver diseases. 2. Molecular mechanisms of definitive endoderm differentiation from human embryonic stem cellsThere is a series of problems to be solved in stem cells-derived hepatocytes differentiation. The most important question is about embryonic stem cells or induced pluripotent stem cells to differentiate into definitive endoderm, and especially the differentiation mechanism of hepatocytes is still far from clear, which greatly limits the efficiency of stem cells-derived hepatocytes differentiation. In this part, we have built up a differentiation model of human embryonic stem cells into definitive endoderm in feeder-free and serum-free conditions. And then, differentially expressed miRNAs between human embryonic stem cells-derived definitive endoderm and undifferentiation hESCs were identified by miRNA-Array screening to get a set of candidate miRNAs which may be involved in definitive endoderm specification. Bioinformatics analysis was then performed to predict the target genes of these candidate miRNAs, which included CTGF. Then, we found that CTGF plays an important role in the self renewal and differentiation of hESCs. At the same time, comparative proteomic analysis of endoderm cells differentiated from hESCs was performed. Proteins with altered expression levels during endoderm differentiation were screened by 2-D DIGE and mass spectrometric analysis. Our results suggest that these proteins could be important epigenetic markers for identifying endoderm cells differentiated from hESCs, and could play important roles in the differentiation of endoderm cells from hESCs or in human endoderm development. These works make significant progress for understanding the molecular mechanisms of hESCs-derived definitive endoderm differentiation.Overall, these studies show that transplantation of encapsulated hepatocytes and human FLSCs/bFGF can improve the liver regeneration in an acute liver failure mouse model, which will ultimately help to provide a new way to treatment the end-stage liver diseases. At the same time, the research on molecular mechanisms of definitive endoderm differentiation from human embryonic stem cells is to lay the foundation for the final establishment of efficient strategy of stem cell differentiation.