Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids

Abstract

The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The ''oval cell'' response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.

Mechano-modulatory synthetic niches for liver organoid derivation

“…A promising alternative to liver transplantation comes from the recent breakthrough that liver organoids can be generated in vitro within animal-derived matrices (e.g. Matrigel) from mouse and human bile duct-derived bi-potential facultative progenitor cells 1,2,11 or primary hepatocytes 4,5 . These organoids are largely composed of progenitor cells that are genetically stable and can be differentiated into functional hepatocyte-like cells, which are able to engraft and increase survival when transplanted in a mouse model of liver disease 1,2 .…”

Organoid Medicine in Hepatology

“…The precise origin of the organoid‐initiating cells has been investigated in mouse models, but it remains elusive in the human liver . Another novel method to generate Hep‐Orgs has recently been reported . Proliferative hepatocytes were isolated from human fetal livers by Percoll gradient centrifugation, imbedded into the Matrigel, and cultured in medium with growth factors.…”

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

“…We picked the knocked-in organoids that grew after transfection based on fluorescence and established clonal lines. First, we successfully targeted a gene exclusively expressed in hepatocyte organoids by virtually all cells, AFP 30 and generated clonal AFP::mNEON lines ( Fig. 5b, Supplementary Fig.…”

“…For generation of human liver ductal organoids, liver biopsies were obtained from patients undergoing surgery at Rotterdam University Medical Center. Isolation, generation and culture of human hepatocytes and human liver ductal organoids were performed as described in 30 isolated, processed and cultured as described previously 34 . Intestinal organoids were cultured in standard culture conditions (ENR) 30 .…”

“…Isolation, generation and culture of human hepatocytes and human liver ductal organoids were performed as described in 30 isolated, processed and cultured as described previously 34 . Intestinal organoids were cultured in standard culture conditions (ENR) 30 . Differentiation was achieved by supplementing ENR medium with DAPT (10 µM, Sigma Aldrich) (for goblet cell enrichment) or by adding DAPT (10 μM, Sigma Aldrich) and the MEK inhibitor PD0325901 (500 nM; Sigma Aldrich) and withdrawing the p38 MAPK inhibitor SB202190, the TGF-β inhibitor A83-01, nicotinamide and Wnt-conditioned medium from the ENR medium (for enteroendocrine cells), as described in 8 .…”