Int J Stem Cells.  2020 Jul;13(2):279-286. 10.15283/ijsc20060.

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

• Mun SJ
• Hong YH
• Ahn HS
• Ryu JS
• Chung KS
• Son MJ

• KMID: 2504340
• DOI: http://doi.org/10.15283/ijsc20060

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.

Keyword

Liver; Organoids; PSCs; Hepatic organoids; Long-term culture

Figure

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  Fig. 1 Generation of hiPSCs-derived hepatic organoids. (A) Schematic diagram of the generation protocol from hiPSCs to hepatic organoid. (B) Representative bright field image of hiPSCs, definitive endoderm, hepatic endoderm, and hepatic organoids. (C) Bright field image of organoids immediately after Matrigel embedding (D0) and in the same field during culture (D1 to D3). (D) Representative immunofluorescence images of the hepatic organoids stained with E-cadherin and ALB.

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  Fig. 2 Long-term culture of hepatic organoids without passaging. (A) Scheme of long-term culture of the organoids. Matrigel-embedded organoids were maintained for four weeks without Matrigel renewal (upper). Organoids were divided at a 1：2 ratio and replated on fresh Matrigel weekly (lower). (B) Morphology of the organoids in the same field from week 1 to 4 without Matrigel renewal (upper) and with Matrigel renewal (lower). (C) mRNA expression levels of ALB, CK18, CK19, and AFP in organoids without Matrigel renewal and with Matrigel renewal weekly. Data are the mean±SEM (n=3) and analyzed by Student’s t-test, *p<0.05 and ***p<0.001.

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  Fig. 3 Long-term expansion of hepatic organoids by serial passaging. (A) Schematic diagram of long-term culture of organoids by passaging (upper). Representative bright field image of hepatic organoids after passaging at day 0, 1, 3 and 7 in the same field (lower). (B) Representative morphology of each passage of hepatic organoids at day 7. (C) Representative morphology of organoids a day after thawing (upper). Cell viability was determined by cell counting with Trypan blue staining before freezing and 12 hours after thawing (lower). Data are the mean±SEM (n=12). (D) Karyotype analysis of the organoids at passage 40 and 50. (E) mRNA expression levels of ALB and AFP at every 10 passages. Data are the mean±SEM (n=3) and analyzed by Student’s t-test, *p<0.05 and ***p<0.001.

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  Fig. 4 Differentiation potential of the long-term expanded hepatic organoids. (A) Schematic diagram of organoid differentiation for further hepatic maturation. Hepatic medium (HM); Expansion medium (EM); and Differentiation medium (DM). (B) mRNA expression levels of ALB, RBP4, and CYP3A4 in HM- or DM-cultured organoids at passage 10. (C) Representative morphology and (D) mRNA expression levels of ALB, RBP4, and CYP3A4 of differentiated hepatic organoids at each indicated passage. Data are the mean±SEM (n=3) and analyzed by Student’s t-test. *p<0.05; **p<0.01; and ***p<0.001.

Reference

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