citations

                    Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth
                

Authors:

Delilah Hendriks , Benedetta Artegiani , Thanasis Margaritis , Iris Zoutendijk , Susana Chuva de Sousa Lopes , Hans Clevers

In:

Source: Nature
Publication Date: (2024)
Issue: 15: 4034

Research Area:

Gastroenterology

Toxicology

Drug Discovery

Cells used in publication:

Hepatocyte, human: Species: human; Tissue Origin: liver

Experiment

Primary human hepatocyte organoid establishment

PHHs were obtained from commercial sources (Lonza, HUCPG and HUCPI; Sigma-Aldrich, MTOXH10002), PHH donors 1-3, 0.3, 28, 1.7 years, respectively). Upon thawing of the cells according to manufacturer’s instructions, cells were seeded into domes of BME in a 2:1 ratio of BME:AdvDMEM/F12+++ (33µl/drop), with 2 drops per well of a 24-well plate. The FH expansion medium as described above for FH organoids was used for transcriptomic experiments to evaluate PHH organoid outgrowth. To optimize PHH organoid growth, different conditions were tested, including supplementation of the FH expansion medium with combinations of 100ng/ml IL6, 20ng/ml IL11, 20ng/ml IL1ß, or 100ng/ml NRG1, and 10 µM cilofexor (GS-9674, FXRa) (MedChemExpress, HY-109083). The optimized PHH expansion medium consisted of AdvDMEM/F12+++ supplemented with 15% RSPO1-conditioned medium, 1xB-27 Supplement Minus VitaminA, 2.5mM nicotinamide, 1.25mM N-acetyl-L-cysteine, 50ng/ml EGF, 50ng/ml HGF, 20ng/ml TGFa, 10nM gastrin, 3µM CHIR-99021, 5µM A83-01, 50µg/ml primocin, 100ng/ml IL6, and 10µM FXRa. This medium was supplemented with 10µM Y-27632 to minimize anoikis during the first few days of culture. From passage 1, 1.5% Noggin-Fc conditioned medium (UPE, N002) was added to this medium for the first 5–8 days after passaging. Upon culture establishment, fibroblast outgrowth was avoided by initial replating of the cells and contaminating outgrowing large and thin-walled duct-like cystic organoids were meticulously manually removed from the culture. Organoids were cultured at 37 ºC and 5% CO2 and passaged using gentle pipetting 1:2 every 10–14 days for the first month, and every 2–3 weeks thereafter. Cultures were regularly tested for mycoplasma and tested negative without exception.

Abstract

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

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