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

Abstract

AbstractHuman 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 cultureBasic 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‐iPSCsSupport Protocol 1: Confirmation of h‐NPC ability to uptake Sendai virus: GFP‐Sendai infectionSupport Protocol 2: Characterization of h‐NPCs amenable to transduction with Sendai particlesSupport Protocol 3: Characterization of h‐iPSCs: Clearance of viral reprogramming vectorsSupport Protocol 4: Preparation of Matrigel‐coated plates