citations

                    Long-Term Expansion of Functional Human Pluripotent Stem Cell-Derived Hepatic Organoids
                

Authors:

Seon Ju Mun, Yeon-Hwa Hong , Hyo-Suk Ahn , Jae-Sung Ryu , Kyung-Sook Chung , Myung Jin Son

In:

Source:
Publication Date: (2020)
Issue: 13: 2

Research Area:

Gastroenterology

Stem Cells

Toxicology

Drug Discovery

Cells used in publication:

Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Culture Media:

Endothelial Cell Growth Medium 2

Hepatocyte Culture Medium

Experiment

Hepatic organoids generation

Human induced pluripotent stem cells (hiPSCs) generated from human foreskin fibroblasts (CRL-2097, the American Type Culture Collection), using a CytoTune^?-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher; A16517), were routinely maintained on a ?-irradiated mouse embryonic fibroblast feeder in iPSCs culture medium (21) at 37?, 5% CO₂. To assess hepatic differentiation, hiPSCs were seeded onto Matrigel^TM (Corning; 354234)-coated dishes supplemented with PSC medium and cultured for 2~3 days until cells reached 90% confluence. The medium was exchanged with RPMI 1640 (Thermo Fisher; 11875093) based 1×B-27 supplement, minus insulin (Thermo Fisher; A1895601) and 100 ng/mL recombinant human activin A (PeproTech; 120-14e). Cells were further incubated for six days to differentiate into definitive endoderm (DE). Cells were differentiated into hepatic endoderm (HE) by treatment with RPMI 1640 based 1×B27 supplement (Thermo Fisher; 17504044), 10 ng/mL basic fibroblast growth factor (bFGF) (PeproTech; 100-18B), and 20 ng/mL recombinant human bone morphogenetic protein (BMP)4 (PeproTech; 120-05ET) under 5% hypoxia for four days. For hepatic maturation, the medium was replaced with Hepatocyte Culture Medium (Lonza; CC-3198) without epidermal growth factor (EGF), mixed with Endothelial Cell Growth Medium-2 (Lonza; CC-3162) in a 1:1 ratio, supplemented with 2.5% fetal bovine serum (RMBIO; FBS-BBT-5XM), 100 nM dexamethasone (Sigma-Aldrich; D4902), 20 ng/mL recombinant human Oncostatin M (R&D system; 295-OM-050), and 10 ng/mL recombinant human hepatocyte growth factor (HGF) (PeproTech; 100-39) for four days under 5% hypoxia, and subsequently under normoxic conditions for a further eight days or more. Arpproximately 25 days after seeding, cyst-shaped 3D organoids were spontaneously generated from 2D monolayers of mature hepatocytes. The organoids, including a few free-floating organoids, were collected and embedded in Matrigel supplemented with Hepatic Medium (HM). These conditions were previously optimized for maintaining functional hepatic organoids (Supplementary Table S1) (21).

Abstract

A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1:2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1:3~1:5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.

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