Transdiferentiation of Gallbladder Epithelial Cells into Hepatocytes In vitro

• Grant number: 23/10642-1
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: February 01, 2024
• End date: February 29, 2028
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Mayana Zatz
• Grantee: Lara Borges Pacheco
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated scholarship(s): 25/08696-1 - Endogenous Hepatic Gene Network Activation in iPSCs via CRISPRa for Efficient Generation of Induced Hepatocytes, BE.EP.DD

Abstract

Liver diseases are responsible for two million deaths worldwide every year and have diverse causes, such as alcohol, viruses, chemical compounds, and genetic factors. Liver transplantation is the only therapy for liver failure, but the scarcity of donors and the need for immunosuppression represent a great obstacle for its implementation. Bioengineering offers an alternative to traditional transplantation, requiring proliferative and functional cellular sources. Primary human hepatocytes (PHH), the gold standard for cellular assays, lose their phenotype rapidly in vitro. Human-induced pluripotent stem cells (iPSCs) are easy to handle in vitro and can differentiate into various tissues, but their production is costly, time-consuming, and inefficient. Additionally, differentiated iPSCs, such as hepatocyte-like cells (HLCs), primarily exhibit a fetal phenotype. Transdifferentiation reduces manufacturing time and tumorigenic risk, presenting a promising alternative for hepatocyte generation. Prevalent transdifferentiation methods convert mouse fibroblasts into intermediate functional hepatocyte-like cells (iHeps) through the ectopic expression of hepatic transcription factors, resulting in limited functionality and an intermediate phenotype. Thus, this project aims to transdifferentiate alternative cell sources, such as Gallbladder Epithelial Cells, into functional hepatocytes in vitro using the CRISPRon system. The functionality of iHeps will be assessed through RT-qPCR, immunofluorescence, and ELISA assays, while their therapeutic potential will be evaluated through transplantation in murine models of hepatic fibrosis.