Engineered Platforms for iPSC-Derived Human Hepatocyte Differentiation and HBV Infection

Hepatitis B virus (HBV) is a global disease and accounts for 80% of virus-related hepatocellular carcinoma. Conducting drug tests on chimpanzees (the only natural animal model of HBV) is prohibitively expensive and severely restricted. Thus, in vitro human liver platforms are urgently needed for HBV studies. Shortages of donor livers and donor-donor variability prevent primary human hepatocytes (PHHs) use for high-throughput drug screening. In contrast, induced pluripotent stem cell (iPSC)-derived human hepatocyte-like cells (HLCs) provide a nearly infinite cell resource with donor-personalized genetic background; therefore, HLCs are an alternative cell source to PHHs to study HBV. However, the current limitations of HLCs are a fetal-like phenotype and exorbitant expenses for large-scale production. Extracellular matrix (ECM) is essential to affect cell functions and guide cell fate through ECM-cell interactions. Electrospun nanofibers mimic ECM topography and stiffness and contain major ECM proteins that have been shown to support PHH functionality in vitro. Thus, the first goal of this thesis is to utilize natural ECM nanofibers to mature HLCs and investigate the roles of topography and stiffness on HLC differentiation. Currently, cutting-edge gene editing technologies allow dynamic and precise cell differentiation and maturation modulation. The second goal of this thesis is to identify key transcription factors (TFs) crucial to modulating HLC differentiation by integrated RNA sequencing and ATAC sequencing analysis. Overexpression or depression of these genes impacts hepatic gene activation and subsequent hepatocyte development. Moreover, TFs and TF-TF networks relevant to the essential hepatic pathways were also explored to improve hepatic function and maturation. In addition to ECM manipulation and gene editing, coculture with stromal cells is beneficial for cell differentiation and development due to paracrine secretions and cell-cell interactions. Previously, the micropatterned coculture (iMPCC) supported higher hepatic functions of HLCs with fibroblasts over 4 weeks. Thus, the third goal of this thesis is to optimize HBV infection in iMPCC by adding small molecules such as cyclic adenosine monophosphate (cAMP) and Janus-kinase inhibitor (JAKi) and then utilize the HBV-infected iMPCC to investigate HBV infection and screen antiviral drugs.