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

Affiliations
  • 1Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
  • 2Department of Functional Genomics, Korea University of Science & Technology (UST), Daejeon, Korea
  • 3Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea

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

  • 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.

  • 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.

  • 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.

  • 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

References

1. Asrani SK, Devarbhavi H, Eaton J, Kamath PS. 2019; Burden of liver diseases in the world. J Hepatol. 70:151–171. DOI: 10.1016/j.jhep.2018.09.014. PMID: 30266282.
Article
2. Yamaguchi T, Matsuzaki J, Katsuda T, Saito Y, Saito H, Ochiya T. 2019; Generation of functional human hepatocytes in vitro: current status and future prospects. Inflamm Regen. 39:13. DOI: 10.1186/s41232-019-0102-4. PMID: 31308858. PMCID: PMC6604181.
Article
3. Underhill GH, Khetani SR. 2017; Bioengineered liver models for drug testing and cell differentiation studies. Cell Mol Gastroenterol Hepatol. 5:426–439.e1. DOI: 10.1016/j.jcmgh.2017.11.012. PMID: 29675458. PMCID: PMC5904032.
Article
4. Clark M, Steger-Hartmann T. 2018; A big data approach to the concordance of the toxicity of pharmaceuticals in animals and humans. Regul Toxicol Pharmacol. 96:94–105. DOI: 10.1016/j.yrtph.2018.04.018. PMID: 29730448.
Article
5. Huang P, Zhang L, Gao Y, He Z, Yao D, Wu Z, Cen J, Chen X, Liu C, Hu Y, Lai D, Hu Z, Chen L, Zhang Y, Cheng X, Ma X, Pan G, Wang X, Hui L. 2014; Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell. 14:370–384. DOI: 10.1016/j.stem.2014.01.003. PMID: 24582927.
Article
6. Kim Y, Kang K, Lee SB, Seo D, Yoon S, Kim SJ, Jang K, Jung YK, Lee KG, Factor VM, Jeong J, Choi D. 2019; Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells. J Hepatol. 70:97–107. DOI: 10.1016/j.jhep.2018.09.007. PMID: 30240598.
Article
7. Touboul T, Hannan NR, Corbineau S, Martinez A, Martinet C, Branchereau S, Mainot S, Strick-Marchand H, Pedersen R, Di Santo J, Weber A, Vallier L. 2010; Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology. 51:1754–1765. DOI: 10.1002/hep.23506. PMID: 20301097.
Article
8. Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA. 2010; Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. 51:297–305. DOI: 10.1002/hep.23354. PMID: 19998274. PMCID: PMC2946078.
Article
9. Sharma A, Sances S, Workman MJ, Svendsen CN. 2020; Multi-lineage human iPSC-derived platforms for disease modeling and drug discovery. Cell Stem Cell. 26:309–329. DOI: 10.1016/j.stem.2020.02.011. PMID: 32142662.
Article
10. Liu C, Oikonomopoulos A, Sayed N, Wu JC. 2018; Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond. Development. 145:dev156166. DOI: 10.1242/dev.156166. PMID: 29519889. PMCID: PMC5868991.
Article
11. Fowler JL, Ang LT, Loh KM. 2020; A critical look: challenges in differentiating human pluripotent stem cells into desired cell types and organoids. Wiley Interdiscip Rev Dev Biol. 9:e368. DOI: 10.1002/wdev.368. PMID: 31746148.
Article
12. Sakabe K, Takebe T, Asai A. 2020; Organoid medicine in hepatology. Clin Liver Dis (Hoboken). 15:3–8. DOI: 10.1002/cld.855. PMID: 32104569. PMCID: PMC7041950.
Article
13. Kuse Y, Taniguchi H. 2019; Present and future perspectives of using human-induced pluripotent stem cells and organoid against liver failure. Cell Transplant. 28(1 Suppl):160S–165S. DOI: 10.1177/0963689719888459. PMID: 31838891. PMCID: PMC7016460.
Article
14. Li M, Izpisua Belmonte JC. 2019; Organoids - preclinical models of human disease. N Engl J Med. 380:569–579. DOI: 10.1056/NEJMra1806175. PMID: 30726695.
Article
15. Akbari S, Arslan N, Senturk S, Erdal E. 2019; Next-generation liver medicine using organoid models. Front Cell Dev Biol. 7:345. DOI: 10.3389/fcell.2019.00345. PMID: 31921856. PMCID: PMC6933000.
Article
16. Hu H, Gehart H, Artegiani B, LÖpez-Iglesias C, Dekkers F, Basak O, van Es J, Chuva de Sousa Lopes SM, Begthel H, Korving J, van den Born M, Zou C, Quirk C, Chiriboga L, Rice CM, Ma S, Rios A, Peters PJ, de Jong YP, Clevers H. 2018; Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell. 175:1591–1606.e19. DOI: 10.1016/j.cell.2018.11.013. PMID: 30500538.
Article
17. Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MM, Ellis E, van Wenum M, Fuchs SA, de Ligt J, van de Wetering M, Sasaki N, Boers SJ, Kemperman H, de Jonge J, Ijzermans JN, Nieuwenhuis EE, Hoekstra R, Strom S, Vries RR, van der Laan LJ, Cuppen E, Clevers H. 2015; Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell. 160:299–312. DOI: 10.1016/j.cell.2014.11.050. PMID: 25533785. PMCID: PMC4313365.
Article
18. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, Zhang RR, Ueno Y, Zheng YW, Koike N, Aoyama S, Adachi Y, Taniguchi H. 2013; Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 499:481–484. DOI: 10.1038/nature12271. PMID: 23823721.
Article
19. Akbari S, Sevinç GG, Ersoy N, Basak O, Kaplan K, Sevinç K, Ozel E, Sengun B, Enustun E, Ozcimen B, Bagriyanik A, Arslan N, Önder TT, Erdal E. 2019; Robust, long-term culture of endoderm-derived hepatic organoids for disease modeling. Stem Cell Reports. 13:627–641. DOI: 10.1016/j.stemcr.2019.08.007. PMID: 31522975. PMCID: PMC6829764.
Article
20. Wu F, Wu D, Ren Y, Huang Y, Feng B, Zhao N, Zhang T, Chen X, Chen S, Xu A. 2019; Generation of hepatobiliary organoids from human induced pluripotent stem cells. J Hepatol. 70:1145–1158. DOI: 10.1016/j.jhep.2018.12.028. PMID: 30630011.
Article
21. Mun SJ, Ryu JS, Lee MO, Son YS, Oh SJ, Cho HS, Son MY, Kim DS, Kim SJ, Yoo HJ, Lee HJ, Kim J, Jung CR, Chung KS, Son MJ. 2019; Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids. J Hepatol. 71:970–985. DOI: 10.1016/j.jhep.2019.06.030. PMID: 31299272.
Article
22. Xia Y, Izpisua Belmonte JC. 2019; Design approaches for generating organ constructs. Cell Stem Cell. 24:877–894. DOI: 10.1016/j.stem.2019.05.016. PMID: 31173717.
Article
23. Holloway EM, Capeling MM, Spence JR. 2019; Biologically inspired approaches to enhance human organoid complexity. Development. 146:dev166173. DOI: 10.1242/dev.166173. PMID: 30992275. PMCID: PMC6503984.
Article
24. Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N, Chang CF, Schiesser J, Aubert P, Stanley EG, Elefanty AG, Miyaoka Y, Mandegar MA, Conklin BR, Neunlist M, Brugmann SA, Helmrath MA, Wells JM. 2017; Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system. Nat Med. 23:49–59. DOI: 10.1038/nm.4233. PMID: 27869805. PMCID: PMC5562951.
Article
25. Quadrato G, Nguyen T, Macosko EZ, Sherwood JL, Min Yang S, Berger DR, Maria N, Scholvin J, Goldman M, Kinney JP, Boyden ES, Lichtman JW, Williams ZM, McCarroll SA, Arlotta P. 2017; Cell diversity and network dynamics in photosensitive human brain organoids. Nature. 545:48–53. DOI: 10.1038/nature22047. PMID: 28445462. PMCID: PMC5659341.
Article
Full Text Links
  • IJSC
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr