Role of decellularized liver matrix on human embryonic stem cell differentiation into functional hepatocytes

Project (M.A., Biological Sciences (Stem Cell)) -- California State University, Sacramento, 2011.Liver disease is one of the leading causes of death worldwide, and over 25,000 people per year die in the U.S. Often the only option available for liver disease patients is liver transplantation, however, liver transplantation is severely limited due to the shortage of donors and donor-recipient matches, especially for children. Hepatocyte transplantation is an alternative for whole-organ transplants and it has been found to improve liver function. Unfortunately, there is limited access to human hepatocytes and transplanted hepatocytes don't survive very long, leading to problems with engraftment. Human embryonic stem cell (hESC) derived hepatocytes have been sought as an alternative to primary hepatocyte transplantation. The goal of my project was to test and improve hESC-derived hepatocyte differentiation protocols in order to obtain high levels of mature hepatocytes in vitro. Since differentiation protocols vary in length, growth factors used, and microenvironment for culturing, variables were tested to establish an improved protocol for mature hepatocytes using a feeder-free system. Specifically, hESCs were induced to form definitive endoderm (DE) before being matured into hepatocytes and whole organ decellularized liver matrix (DLM) was examined as a carrier for hepatocyte transplantation.\ud The results showed that the differentiated hESC cells had the characteristic polygonal hepatocyte morphology and were able to store glycogen. These differentiated cells secreted albumin and synthesized urea by the end of the protocol (peaking at day 26) which is characteristic of mature hepatocytes. The decellularization of the liver was improved by making it shorter than most decellularization processes and the residual DNA in the DLM was equivalent to other protocols. The gross extracellular matrix shape and structure of the DLM was retained, as shown by immunohistochemistry staining of key ECM proteins: laminin, fibronectin and collagen IV. The effects of DLM on hESC derived hepatocyte survival were analyzed by infusing these cells into the DLM after decellularization. Our data suggests that the DLM improves cell survival in vitro. The DLM also supported cell survival in vivo for up to 7 days in an immunodeficient mouse model. Furthermore, DLM seemed to be more effective in helping fetal hepatocyte cell engraftment in vivo, as compared to splenic injections or Matrigel encapsulation, for up to 8 weeks, as evidenced by bioluminescence data. In addition, hESC derived hepatocytes were examined to determine which integrins were expressed since this would inform us as to their potential interactions with extracellular matrix proteins. It was found that the cells expressed integrins ???1, ???3, ???4, ???5, ???V, ???1, and ???3. This suggests that these cells are capable of responding to extracellular matrix proteins, including collagen, laminin and fibronectin. The identification of these specific integrins provides a basis for understanding the mechanisms behind the effects of liver ECM 3-D culture environment on hESC derived hepatocyte maturation. \ud In conclusion, this study resulted in an improved hESC-derived hepatocyte differentiation protocol and established a short and efficient decellularization protocol for liver decellularization in rodents. The DLM seems to help maintain hepatocyte differentiation in vitro, and it facilitated cell engraftment and long-term survival in vivo when seeded with fetal hepatocytes. Overall, our data suggests that DLM may be developed as an alternative carrier for hepatocyte transplantation.Biological Sciences (Stem Cell