High-Throughput Identification of Molecular Factors that Promote Phenotypic Stabilization of Primary Human Hepatocytes in vitro
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CitationShan, Jing. 2016. High-Throughput Identification of Molecular Factors that Promote Phenotypic Stabilization of Primary Human Hepatocytes in vitro. Doctoral dissertation, Harvard Medical School.
AbstractLiver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation, but their development has been hindered by the rapid loss of liver-specific functions in cultured hepatocytes. The overall goal of this thesis is to systematically identify genetic factors involved in hepatocyte phenotype maintenance in vitro in order to help generate a source of functional human hepatocytes for studying liver biology and treating liver disease. It is our hypothesis that molecular signals from the stroma provide inductive cues to maintain liver phenotype and that these stromal signals can be isolated and used to stabilize primary human hepatocytes in vitro. We report here the development of a high-throughput human liver model and attendant automatable assays capable of reflecting human liver physiology. These tools were used to conduct genetic knock down screens of over 450 stromal factors in over 5000 two-way combinations in order to identify molecules important for hepatocyte functions. Results suggest that multiple signaling molecules are involved in stromal-mediated stabilization of hepatocytes ex vivo. Adsorption of hit molecules such as Activin A onto tissue culture plastic improved hepatocyte survival and morphology, and may be acting via signaling pathways that inhibit cell cycle progression and apoptosis. These results represent important first steps in the elucidation of mechanisms instrumental to the functional maintenance of hepatocytes in vitro, and we hope this new insight will guide the assembly of a cocktail of recombinant acellular stromal products capable of replacing stromal cells in hepatic tissue engineering applications.
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