Endogenous Hepatic Gene Network Activation in iPSCs via CRISPRa for Efficient Generation of Induced Hepatocytes

• Grant number: 25/08696-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: September 01, 2025
• End date: August 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Mayana Zatz
• Grantee: Lara Borges Pacheco
• Supervisor: Charles Gersbach
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Duke University, United States
• Associated to the scholarship: 23/10642-1 - Transdiferentiation of Gallbladder Epithelial Cells into Hepatocytes In vitro, BP.DD

Abstract

Liver failure represents a global health crisis, with limited treatment options due to the shortage of donor organs. Alternatives like hepatocyte transplantation and bioartificial livers depend on functional, expandable hepatocytes, but existing cell models fail to preserve liver-specific functions in vitro. Human induced pluripotent stem cells (hiPSCs) can be differentiated into hepatocyte-like cells using chemical-based protocols, but these methods are time-consuming, costly, and yield cells with a fetal phenotype. Transcription factor-based approaches to induce hepatocyte fate offer a promising solution, yet current protocols rely on the exogenous expression of key liver transcription factors (TFs), often resulting in immature, intermediate phenotypes. In this project, we propose a novel strategy to generate induced hepatocytes (iHeps) by activating the endogenous liver gene regulatory network (GRN) in iPSCs using CRISPRa. By targeting TFs identified through CRISPRa-based screening, we aim to reprogram iPSCs into hepatocyte-like cells. To further enhance cellular maturation, we will incorporate non-transcription factor regulators such as UBR5 and miRNA-122. We will use transcriptional and epigenetic profiling (RNA-seq and ATAC-seq) to assess the activation of liver-specific GRNs, chromatin remodeling, and cellular maturation. Our findings will provide valuable insights into optimizing reprogramming strategies for generating iHeps, potentially fostering therapeutic applications for liver transplantation. (AU)