Functional hepatobiliary organoids recapitulate liver development and reveal essential drivers of hepatobiliary cell fate determination

Abstract

During liver development, hepatocytes, and cholangiocytes are concurrently differentiated from common liver progenitor cells and are assembled into hepatobiliary architecture to perform proper hepatic function. However, the generation of functional hepatobiliary architecture from hepatocytes in vitro is still challenging, and the exact molecular drivers of hepatobiliary cell lineage determination is largely unknown. In this study, functional hepatobiliary organoids (HBOs) are generated from hepatocytes. These HBOs contain a bile duct network surrounded by mature hepatocytes and stably maintain hepatic characteristics and function in vitro and upon transplantation in vivo. Morphological transition and expression profile of hepatocyte-derived organoids recapitulate the process of liver development. Gene regulation landscape of hepatocyte-derived organoids reveal that Tead4 and Ddit3 promote the cell fate commitment of liver progenitors to functional cholangiocytes and hepatocytes, respectively. Liver cell fate determination is reversed by inhibiting Tead4 or increasing Ddit3 expression both in vitro and upon transplantation in vivo. Collectively, hepatocyte-derived HBOs reveal the essential transcription drivers of liver hepatobiliary cell lineage determination and represent powerful models for liver development and regeneration.

Keywords

liver development / cell lineage plasticity / hepatobiliary organoids

References

[1] Yimlamai D, Christodoulou C, Galli GG, et al. Hippo pathway activity influences liver cell fate. Cell 2014;157:1324–38.

[2] Tarlow BD, Carl P, Naugler Willscott E, et al. Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes. Cell Stem Cell 2014;15:605–18.

[3] Schaub JR, Huppert Kari A, Kurial Simone NT, et al. De novo formation of the biliary system by TGFbeta-mediated hepatocyte transdifferentiation. Nature 2018;557:247–51.

[4] Yanger K, David K, Yiwei Z, et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 2014;15:340–49.

[5] Wang B, Zhao L, Fish M, et al. Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver. Nature 2015;524:180–5.

[6] Pu W, Zhang H, Huang X, et al. Mfsd2a+ hepatocytes repopulate the liver during injury and regeneration. Nat Commun 2016;7:13369.

[7] Lin S, Nascimento Elisabete M, Gajera Chandresh R, et al. Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury. Nature 2018;556:244–8.

[8] Raven A, Wei-Yu L, Man Tak Y, et al. Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration. Nature 2017;547:350–4.

[9] Deng X, Zhang X, Li W, et al. Chronic liver injury induces conversion of biliary epithelial cells into hepatocytes. Cell Stem Cell 2018;23:114–22.e3.

[10] Zaret KS and Grompe M. Generation and regeneration of cells of the liver and pancreas. Science 2008;322:1490–4.

[11] Squires RH, Vicky N, Rene R, et al. Evaluation of the pediatric patient for liver transplantation: 2014 practice guideline by the American Association for the Study of Liver Diseases, American Society of Transplantation and the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. Hepatology 2014;60:362–98.

[12] Knouse KA, Lopez KE, Bachofner M, et al. Chromosome segregation fidelity in epithelia requires tissue architecture. Cell 2018;175:200–11.e13.

[13] Zhu S, Milad R, Jack H, et al. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 2014;508:93–7.

[14] Huang P, Ludi Z, Yimeng G, et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 2014;14:370–84.

[15] Si-Tayeb K, Noto Fallon K, Masato N, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010;51:297–305.

[16] Rashid ST, Corbineau S, Hannan N, et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest 2010;120:3127–36.

[17] Ma X, Yuyou D, Benjamin T-S, et al. Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells. Stem Cells Transl Med 2013;2:409–19.

[18] Liu H, Kim Y, Sharkis S, et al. In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins. Sci Transl Med 2011;3:82ra39.

[19] Basma, H, Alejandro S-G., Rao YG, et al. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 2009;136:990–9.

[20] Woo DH, Suel-Kee K, Hee-Joung L, et al. Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice. Gastroenterology 2012;142:602–11.

[21] Katsuda T, Masaki K, Keitaro H, et al. Conversion of terminally committed hepatocytes to culturable bipotent progenitor cells with regenerative capacity. Cell Stem Cell 2017;20:41–55.

[22] Wu H, Xu Z, Gong-Bo F, et al. Reversible transition between hepatocytes and liver progenitors for in vitro hepatocyte expansion. Cell Res 2017;27:709–12.

[23] Kim Y, Kang K, Lee SB, et al. Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells. J Hepatol, 2018.

[24] Unzu C, Planet E, Brandenberg N, et al. Pharmacological induction of a progenitor state for the efficient expansion of primary human hepatocytes. Hepatology 2019;69:2214–31.

[25] Sampaziotis F, de Cardoso BM, Pedro M, et al. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat Biotechnol 2015;33:845–52.

[26] Mehrian-Shai R, Chen CD, Shi T, et al. Insulin growth factor-binding protein 2 is a candidate biomarker for PTEN status and PI3K/Akt pathway activation in glioblastoma and prostate cancer. Proc Natl Acad Sci U S A 2007;104:5563–8.

[27] Han X, Wang Y, Pu W, et al. Lineage tracing reveals the bipotency of SOX9(+) hepatocytes during liver regeneration. Stem Cell Rep 2019;12:624–38.

[28] Sato T, Vries Robert G, Snippert Hugo J, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459:262–5.

[29] Eiraku, M, Nozomu T, Hiroki I, et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 2011;472:51–6.

[30] Takasato M, Er Pei X, Chiu Han S, et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 2015;526:564–8.

[31] Takebe T, Keisuke S, Masahiro E, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013;499:481–4.

[32] He J, Xiaoyu Z, Xinyi X, et al. Organoid technology for tissue engineering. J Mol Cell Biol 2020;12:569–79.

[33] Aloia L, McKie MA, Vernaz G, et al. Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regeneration. Nat Cell Biol 2019;21:1321–33.

[34] Huch M, Helmuth G, Ruben VB, et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 2015;160:299–312.

[35] Gordillo M, Evans T, Gouon-Evans V. Orchestrating liver development. Development 2015;142:2094–108.

[36] Yang L, Wang WH, Qiu WL, et al. A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation. Hepatology 2017;66:1387–401.

[37] Stadhouders R, Filion GJ, Graf T. Transcription factors and 3D genome conformation in cell-fate decisions. Nature 2019;569:345–54.

[38] Peng G, Shengbao S, Guizhong C, et al. Molecular architecture of lineage allocation and tissue organization in early mouse embryo. Nature 2019;572:528–32.

[39] Zong Y, Stanger BZ. Molecular mechanisms of liver and bile duct development. Wiley Interdiscip Rev Dev Biol 2012;1:643–55.

[40] Planas-Paz L, Tianliang S, Monika P, et al. YAP, but Not RSPOLGR4/5, signaling in biliary epithelial cells promotes a ductular reaction in response to liver injury. Cell Stem Cell 2019;25:39–53.e10.

[41] Camp JG, Keisuke S, Tobias G, et al. Multilineage communication regulates human liver bud development from pluripotency. Nature 2017;546:533–8.

[42] Wu F, Wu D, Ren Y, et al. Generation of hepatobiliary organoids from human induced pluripotent stem cells. J Hepatol, 2019.

[43] Vyas D, Baptista PM, Brovold M, et al. Self-assembled liver organoids recapitulate hepatobiliary organogenesis in vitro. Hepatology, 2017.

[44] Tanimizu N, Ichinohe N, Sasaki Y, et al. Generation of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo. Nat Commun 2021;12:3390.

[45] Spence JR, Lange Alex W, Lin Suh-Chin J, et al. Sox17 regulates organ lineage segregation of ventral foregut progenitor cells. Dev Cell 2009;17:62–74.

[46] Parviz F, Christine M, Garrison Wendy D. et al. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis. Nat Genet 2003;34:292–6.

[47] Jiang Y, Dechun F, Xiaochao M, et al. Pregnane X receptor regulates liver size and liver cell fate by yes-associated protein activation in mice. Hepatology 2019;69:343–58.

[48] Antoniou A, Raynaud P, Cordi S, et al. Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. Gastroenterology 2009;136:2325–33.

[49] Schrem H, Klempnauer J, Borlak J. Liver-enriched transcription factors in liver function and development. Part II: the C/EBPs and D site-binding protein in cell cycle control, carcinogenesis, circadian gene regulation, liver regeneration, apoptosis, and liver-specific gene regulation. Pharmacol Rev 2004;56:291–330.

[50] Azuma H, Nicole P, Aarati R, et al. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice. Nat Biotechnol 2007;25:903–10.

[51] Gao D, Vela I, Sboner A, et al. Organoid cultures derived from patients with advanced prostate cancer. Cell 2014;159:176–87.

[52] Dekkers JF, Alieva M, Wellens LM, et al. High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc 2019;14:1756–71.

[53] Fellmann C, Hoffmann T, Sridhar V, et al. An optimized microRNA backbone for effective single-copy RNAi. Cell Rep 2013;5: 1704–13.

[54] Corces MR, Trevino Alexandro E, Hamilton Emily G, et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 2017;14:959–62.

[55] Palmer NP, Schmid PR, Berger B, et al. A gene expression profile of stem cell pluripotentiality and differentiation is conserved across diverse solid and hematopoietic cancers. Genome Biol 2012;13:R71.

[56] Ben-Porath I, Thomson Matthew W, Carey Vincent J, et al. An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 2008;40:499–507.

[57] Lawrence,MG, Taylor Renea A, Roxanne T, et al. Australian Prostate Cancer BioResource. A preclinical xenograft model of prostate cancer using human tumors. Nat Protoc 2013;8:836–48.

RIGHTS & PERMISSIONS

The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.