Reprogramming of Human Hepatic Non-Parenchymal Cells: Step-by-Step Protocol

Abstract

Human induced pluripotent stem cells (h-iPSCs) represent a potentially unlimited source for the generation of human hepatocyte-like cells (h-iHLCs) for the establishment of platforms to study drug-induced hepatotoxicity, liver disease modeling, and ultimately the application of h-iHLCs in treatment of patients with end-stage liver disease. To understand the impact of donor-specific factors on the generation of h-iHLCs, the model for the direct comparison of h-iHLCs and primary human hepatocytes (PHHs) from the same human donor is needed. This study proposes a step-by-step protocol for plating, expansion, and characterization of primary human hepatic non-parenchymal cells (h-NPCs) isolated from the human liver, the reprogramming of generated h-NPCs into h-iPSCs and subsequent differentiation of reprogrammed h-iPSCs into h-iHLCs. The ultimate goal is to compare the gene expression involved in hepatocyte metabolism between h-iHLCs and PHHs from the same human donor thus eliminating interdonor variability. This newly developed protocol of h-NPC culture, expansion, and reprogramming into h-iPSCs allows: (1) utilization of a single organ source (i.e., liver) for isolation of PHHs and h-NPCs and (2) the in-depth study of donor factors involved in the generation of h-iHLCs with direct comparison to PHHs from the same donor. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Plating and expansion of human hepatic NPCs in culture

Basic Protocol 2: Reprogramming of h-NPCs to h-NPC-derived induced pluripotent stem cells (h-iPSCs)

Basic Protocol 3: Culture, passaging, and freezing of h-iPSCs

Support Protocol 1: Confirmation of h-NPC ability to uptake Sendai virus: GFP-Sendai infection

Support Protocol 2: Characterization of h-NPCs amenable to transduction with Sendai particles

Support Protocol 3: Characterization of h-iPSCs: Clearance of viral reprogramming vectors

Support Protocol 4: Preparation of Matrigel-coated plates