Generation of multi-cellular human liver organoids from pluripotent stem cells

Abstract

A growing number of in vitro hepatic models exist to study human genetics, liver biology, disease modeling and drug development and range from 2D hepatocytes to 3D multi-cellular tissues that are derived from human stem cells. However, stem cell-based models generally suffer from batch-, clone- and donor-dependent variability, hindering broader usage in diverse biomedical applications. To circumvent this challenge, we herein describe a reproducible protocol to generate human liver organoids in 20-25 days derived from pluripotent stem cells (PSCs). These organoids are intra-luminally polarized to form canalicular structures and are comprised of mainly hepatic epithelial cells, co-differentiated with stellate-like and hepatic macrophage-like cells that enables hepatic inflammatory disease modeling in vitro. These multi-lineage liver organoids express hepatocyte genes, secrete albumin and have vital metabolic functions. This protocol utilizes PSC derived 3D human liver organoids as a renewable, reproducible and personalized cell source, thus facilitating disease modeling and mechanistic studies with a future goal of developing novel therapeutics against currently intractable diseases.

Keywords: Human liver organoids; Pluripotent stem cells; Self-organization; Steatohepatitis.

Figure 1.

Schematic of liver organoid formation. This method follows a differentiation sequence that goes through DE and then an early gut stage. Clusters or clumps of cells are then embedded into Matrigel. The protocol ends with treatment of complete HCM and liver organoids at day 20-25.

Figure 2.

Successful co-differentiation is critically dependent on the starting cell density. A. top shows an optimal cell density of about 90% confluence, the middle panel shows a low cell density, and the lower panel is overly confluent. B. On day 6-7 spheroids will form as 3D structures attached to the monolayer and floating in the media.

Figure 3.

Visualization of liver organoid formation morphology in Matrigel over time. A) Day 8, B) Day 12, C) Day 15, D) Day 21.

Figure 4.

Whole mount staining of liver organoids, day 23. Representative images of A. HNF4-α (red), B. E-cadherin (green), C. DAPI (white), D. ZO-1 (purple), and merge.

Figure. 5.

OA treatment of liver organoids, day 23. A,C, are phase contrast images of before and after treatment of liver organoids with OA. B,D are live confocal images before and after treatment with OA showing lipid accumulation within an organoid. F-actin (red) is a membrane stain, DAPI (blue) is a nuclear stain, and BODIPY (green) is a lipid stain.