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Generation of Functional Human Hepatocytes in Vitro: Current Status Yamaguchi et al. Inflammation and Regeneration (2019) 39:13 Inflammation and Regeneration https://doi.org/10.1186/s41232-019-0102-4 REVIEW Open Access Generation of functional human hepatocytes in vitro: current status and future prospects Tomoko Yamaguchi1,2, Juntaro Matsuzaki2,3, Takeshi Katsuda2, Yoshimasa Saito1, Hidetsugu Saito1 and Takahiro Ochiya2,4* Abstract Liver and hepatocyte transplantation are the only effective therapies for late-stage liver diseases, in which the liver loses its regenerative capacity. However, there is a shortage of donors. As a potential alternative approach, functional hepatocytes were recently generated from various cell sources. Analysis of drug metabolism in the human liver is important for drug development. Consequently, cells that metabolize drugs similar to human primary hepatocytes are required. This review discusses the current challenges and future perspectives concerning hepatocytes and hepatic progenitor cells that have been reprogrammed from various cell types, focusing on their functions in transplantation models and their ability to metabolize drugs. Keywords: Hepatocyte, Regeneration, Progenitor cells, Drug metabolism Background underlying liver carcinogenesis, and to assist the devel- The prognosis of patients with end-stage liver cirrhosis opment of personalized therapies for patients with hepa- and fulminant hepatitis is poor unless they receive a liver tocellular carcinoma. This review discusses the current transplant [1]. Unfortunately, there is a shortage of challenges associated with therapeutically relevant ap- transplantable organs, and consequently, alternatives proaches for regenerating hepatocytes in vitro and future have been explored. Although the resected human liver perspectives for hepatocytes and hepatic progenitor cells has an enormous regenerative capacity [2], the functions reprogrammed from various cell types. Particular focus of primary human hepatocytes decrease upon conven- is paid to the functions of these cells in transplantation tional two-dimensional culture on an extracellular models and their ability to metabolize drugs. matrix-coated surface. Functional human hepatocytes can be generated in vitro due to recent technological ad- Main text vances in the stem cell research field [3]. This approach Animal models for hepatocyte transplantation could be an abundant source of cells for therapeutic ap- experiments plications. In addition, in vitro culture of human hepato- Evaluation of the repopulation rate and hepatic function cytes and/or their progenitors may help to increase of transplanted human primary hepatocytes has in- understanding of liver development and regeneration creased over the past two decades with the development following injury, to estimate the risk of drug-induced of various mouse models (Table 1). There are three main liver injury, to analyze the interactions between hepato- mouse models: albumin (ALB) uroplasminogen activator cytes and hepatitis virus, to elucidate the mechanisms (uPA) transgenic mice, mice with knockout of the fumarylacetoacetate hydrolase (Fah) gene, and ALB thy- * Correspondence: [email protected] midine kinase transgenic-NOD-SCID-interleukin com- 2Division of Molecular and Cellular Medicine, National Cancer Center mon mice gamma chain knockout (TK-NOG) mice [19]. Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan In uPA/SCID mice, constitutive expression of uPA in 4Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan hepatocytes causes liver injury and permits selective ex- Full list of author information is available at the end of the article pansion of transplanted human hepatocytes. However, © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yamaguchi Table 1 Comparison of potential cell sources for cell-based treatment of liver failure Cell source Methods Features CYP positivity Animal model Repopulation efficiency Serum human ALB Ref. and activity in concentration vitro et al. Inflammation and Regeneration DE cells iPSCs Activin A Prolonged survival – NSG mice treated with 13–35% – [4] DMN (chronic liver injury) ICGhigh HL ESCs Lithium, OSM, DEX, and HGF Gamma-glutamyl transpeptidase Expression of BALB/c nude mice treated 10.2 ± 3.11% at 7 weeks 3 μg/ml at 7 [5] cells activity, glycogen accumulation, CYP1A2 and with CCl4 (acute liver injury) weeks and urea secretion CYP3A4 iPSC-LBs iPSCs Coculture with endothelial and Early hepatic marker positive and Expression of TK-NOG mice – 1.7 μg/ml at 6 [6] mesenchymal cells connections between human and CYP3A7 weeks host vessels HL cells ESCs/iPSCs Activin A, FGF, HGF, and DEX LDL uptake, lipid storage, Expression of MUP-uPA/SCID/Bg mice 1–20% at 100 days 0.1–6.4 mg/ml at [7] glycogen storage, and uptake and CYP3A4 100 days excretion of ICG (2019)39:13 iMPC- Fibroblasts OCT4, SOX2, KLF4, CHIR99021, Hepatocyte marker positive Activities of FRG mice 2% at 9 months 100 μg/ml at ~ 35 [8] Heps DLPC, NaB, Par, RG, Activin A, CYP3A4 and weeks bFGF, EGF, A83-01, BMP4, DEX, CYP2C19 HGF, OSM, and Compound E − − − − iHeps Fibroblasts HNF6, HNF4α, HNF1α, CEBPA, Glycogen synthesis, LDL uptake, Activities of Tet-uPA/Rag2 / /γc / mice 30% at 7 weeks 150 μg/mL at 7 [9] PROX1, and ATF5 exclusion of absorbed ICG, and CYP3A4, weeks accumulation of fatty droplets CYP1A2, and CYP2B6 iHeps/ Fibroblasts FOXA3, HNF1β, and HNF4α (SV40) Hepatocyte marker positive, Activities of F/R mice 0.3–4.2% at 9 weeks 350 ng/ml at 9 [10] iHepsLT glycogen storage, ICG absorption, CYP1A2, weeks acetylated LDL uptake, and CYP2A, and cytoplasmic accumulation of CYP2D6 triglycerides and lipids hiEndoPC- Gastric Bay, Bix, RG, SB, BMP4, Wnt3a, Uptake of ICG and LDL, glycogen CYP3A4 F/R mice 10% at 8–10 weeks 350 ng/ml at 8 [11] Heps epithelial cells FGF4, HGF, DEX, and OSM storage, and accumulation of activity weeks fatty droplets CFPHs Hepatocytes Long-term culture Liver progenitor cell marker Expression of uPA/SCID mice 0.2–27.0% at 9–10 weeks 9–728 μg/mL at [12, positive CYP2C9, 9–10 weeks 13] CYP2C19, CYP1A1, and CYP1A2 Human Ductal cells N2, B27, N-acetylcysteine, gastrin, Hepatocyte morphology, CYP3A4 BALB/c nude mice treated – 80 ng/ml at 6 [14] liver (EPCAM+) EGF, R-spondin1, FGF10, HGF, glycogen accumulation, and LDL activity with CCl4 (acute liver weeks organoid nicotinamide, A83-01, and FSK uptake injury) hCdHs Hepatocytes HGF, A83-01, and CHIR99021 High nucleus-to-cytoplasm ratio, CYP1A2 Alb-TRECK/SCID mice – 1.5 μg/ml [15] pluripotency stem cell marker activity positive, and hepatic progenitor cell marker expression Page 2 of 9 HepLPCs Hepatocytes N2, B27, HGF, EGF, Y27632, A83- High nucleus-to-cytoplasm ratio Activities of FRG mice 13% – [16] 01, CHIR99021, S1P, and LPA and liver progenitor cell marker CYP1A2, expression CYP2B6, and Table 1 Comparison of potential cell sources for cell-based treatment of liver failure (Continued) Yamaguchi Cell source Methods Features CYP positivity Animal model Repopulation efficiency Serum human ALB Ref. and activity in concentration vitro et al. Inflammation and Regeneration CYP3A4 ProliHHs Hepatocytes Wnt3a, N2, B27, N-acetylcysteine, Progenitor-associated gene CYP2B6 FRG mice 64 ± 21.8% (at P4) at 4 5.8 ± 4.5 mg/ml (at [17] gastrin, EGF, FGF10, HGF, expression and biphenotypic cells activity months P4) at 4 months nicotinamide, A83-01, FSK, and Y27632 Hep-Orgs Fetal and RSPO1 conditioned medium, B27, Networks of bile canaliculi, PAS CYP3A4 FNRG mice – 200 μg/ml after [18] cryopreserved EGF, N-acetylcysteine, gastrin, staining, and LDL uptake activity and 90 days primary CHIR99021, HGF, FGF7, FGF10, CYP2E1 hepatocytes A83-01, nicotinamide, Rho inhibi- expression tor g-27,632, and TGFα (2019)39:13 Page 3 of 9 Yamaguchi et al. Inflammation and Regeneration (2019) 39:13 Page 4 of 9 uPA/SCID mice have some disadvantages. Repopulation regenerate the injured liver in mice, which indicates the of human hepatocytes in the liver of these mice is de- feasibility of mouse models for checking the function of creased due to deletion of the uPA transgene by hom- in vitro-derived cells. ologous recombination. In addition, hemizygotes cannot be used as hosts because homologous recombination oc- Potential alternative cell sources for hepatocyte curs more frequently in hemizygotes than in homozy- transplantation therapy gotes. To overcome these disadvantages, Tateno et al. To overcome the shortage of donor hepatocytes, many established a novel host strain that expresses a transgene attempts have been made to generate functional hepato- comprising the ALB promoter/enhancer and uPA cDNA cytes from multiple types of cells (Table 1). However, and is of a SCID background (cDNA-uPA/SCID mice) there is controversy regarding the usefulness of these [20]. Tesfaye et al. also generated a novel mouse strain cells for transplantation therapy. Liu et al. generated hu- that expresses the uPA gene under the control of the man induced pluripotent stem cell (iPSC) lines
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