Children are particularly susceptible to adverse effects of drugs and environmental chemicals due to their immature ability to process xenobiotics. Supported by the parent R01 grant, the investigators are actively studying developmental regulation of drug processing genes (DPGs) in mice. Their long-term goal is to understand species differences and similarities in xenobiotic metabolism and hepatotoxicity, a huge challenge in drug development and risk assessment of human exposure to environmental chemicals. The objective of this proposal is to recruit new collaborators with expertise in stem-cell/cancer research, bioengineering, and bioinformatics to establish a transdisciplinary team and a virtual consortium to develop a novel 3D culture model of hepatocyte differentiation and maturation from stem cells to bridge the huge gap in translating scientific findings from animals to humans. The current bottleneck in hepatocytes differentiation from stem cells in vitro is that the differentiated hepatocytes are largely immature hepatocytes with very low expression of DPGs, which are required to evaluate xenobiotic metabolism and hepatotoxicity. 3D culture provides a microenvironment essential for cell differentiation and hepatocyte function. Growth hormone (GH) plays a key role in postnatal liver development. Oxygen availability is essential in differentiation of stem cells and metabolic function of hepatocytes. Hypoxia-inducible factor 1a (HIF-1?) and NF-?B are master regulators of hypoxic response. Hepatocyte nuclear factor 4? (HNF4?) is a master regulator of hepatocyte differentiation and liver function. ?-catenin plays a key role in not only liver morphogenesis, but also hepatic basal expression and induction of DPGs. There are extensive interactions among HIF-1?, NF-?B, HNF4?, and ?-catenin in gene regulation; however, how they interact and regulate hepatocyte differentiation and maturation during liver development is unknown. The central hypothesis is that 3D microenvironment, oxygen availability, and GH are essential in hepatocyte maturation via affecting interactions among HIF-1?, NF-?B, HNF4?, Stat5, ?-catenin, and the ?-catenin effector LEF-1. This central hypothesis will be tested in 3 specific aims.
Aim 1 will use ChIP- sequencing to determine the interactions among HIF-1?, NF-?B, HNF4?, Stat5, ?-catenin, and LEF-1 in regulating gene expression during hepatocyte differentiation and maturation in mouse liver and a 3D culture model.
Aim 2 will determine the physical interaction between HNF4? and LEF-1/TCF4 and effects of ?-catenin modulators on the differentiation and maturation of hepatocytes from mouse stem cells.
Aim 3 will determine effects of a 3D nanofibrous scaffold, oxygen tension, and GH on the maturation of hepatocytes differentiated from mouse stem cells. This novel 3D culture model of hepatocyte differentiation and maturation from stem cells will be an invaluable tool to elucidate species differences and similarities between humans and mice in: 1) developmental regulation of DPGs; 2) effects of environmental chemicals and therapeutic drugs on hepatocyte differentiation and maturation during liver development; and 3) metabolism and hepatotoxicity of xenobiotics.
The proposed studies are of great importance in an under-investigated area of regulatory mechanisms of the developmental expression patterns of genes important in the absorption; distribution; metabolism; and excretion of therapeutic drugs. Thus; the findings are expected to be applicable to the improvement of efficacy and safety of pediatric pharmacology. Results from this study will set a solid foundation for future comparative studies on species differences and similarities between humans and mice in developmental regulation of drug processing genes as well as metabolism and hepatotoxicity of xenobiotics; which are huge challenges in drug development and risk assessment of human exposure to environmental chemicals.
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