Background: Induced pluripotent stem cells (iPSCs) are human somatic cells that have been reprogrammed to a pluripotent state. Several methods have been used to generate hepatocyte-like cells from iPSCs. However, these hepatic cells have limited clinical application because of their immature function compared to primary hepatocytes. Mesenchymal stem cells (MSCs) have been reported to inhibit apoptosis of hepatic cells and to improve hepatic regeneration in acute liver injury. Therefore, we expected that MSCs had the potential to positively contribute to the maturation of hepatic cells. Here we demonstrate the effect of MSCs on the maturation of hepatoblasts derived from human iPSCs. Methods: MSCs were isolated from human bone marrow and cultured to 70-80% confluence. MSC-conditioned medium (MSC-CM) was collected 48 h after culture in hepatic maturation medium. Human iPSC-derived hepatoblasts were then cultured for 6 days with MSC-CM. Hepatic functions were analyzed and compared to those from cells cultured in general maturation medium. Results: Cells in both groups had a cuboidal morphology typical of hepatocytes. The proportion of Oct4-positive cells was decreased and those of albumin- and alpha-fetoprotein-positive cells were increased in the MSC-CM group. Albumin secretion and urea synthesis as well as cytochrome P450 (CYP) 3A4 activity were enhanced in the MSC-CM group. The gene expressions of some CYP enzymes were upregulated as demonstrated by RT-PCR. Conclusion: Secreted molecules from human MSCs could enhance the hepatic function of human iPSC-derived hepatocyte-like cells. Although more technological innovations are needed, MSC-CM will be useful as a novel efficient strategy for clinically relevant hepatic cell maturation.

1.
Collin de l'Hortet A, Takeishi K, Guzman-Lepe J, Handa K, Matsubara K, Fukumitsu K, et al: Liver-regenerative transplantation: regrow and reset. Am J Transplant 2016;16:1688-1696.
2.
Greenhough S, Medine CN, Hay DC: Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening. Toxicology 2010;278:250-255.
3.
Yu Y, Liu H, Ikeda Y, Amiot BP, Rinaldo P, Duncan SA, et al: Hepatocyte-like cells differentiated from human induced pluripotent stem cells: relevance to cellular therapies. Stem Cell Res 2012;9:196-207.
4.
Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, et al: Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010;51:297-305.
5.
Takayama K, Inamura M, Kawabata K, Sugawara M, Kikuchi K, Higuchi M, et al: Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1α transduction. J Hepatol 2012;57:628-636.
6.
Nagamoto Y, Tashiro K, Takayama K, Ohashi K, Kawabata K, Sakurai F, et al: The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets. Biomaterials 2012;33:4526-4534.
7.
Yagi H, Tafaleng E, Nagaya M, Hansel MC, Strom SC, Fox IJ, et al: Embryonic and induced pluripotent stem cells as a model for liver disease. Crit Rev Biomed Eng 2009;37:377-398.
8.
Chamberlain G, Fox J, Ashton B, Middleton J: Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 2007;25:2739-2749.
9.
Christ B, Stock P: Mesenchymal stem cell-derived hepatocytes for functional liver replacement. Front Immunol 2012;3:168.
10.
Vemuri MC, Chase LG, Rao MS: Mesenchymal stem cell assays and applications. Methods Mol Biol 2011;698:3-8.
11.
Sgodda M, Aurich H, Kleist S, Aurich I, König S, Dollinger MM, et al: Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res 2007;313:2875-2886.
12.
Stock P, Brückner S, Ebensing S, Hempel M, Dollinger MM, Christ B: The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver. Nat Protoc 2010;5:617-627.
13.
Yagi H, Parekkadan B, Suganuma K, Soto-Gutierrez A, Tompkins RG, Tilles AW, et al: Long-term superior performance of a stem cell/hepatocyte device for the treatment of acute liver failure. Tissue Eng Part A 2009;15:3377-3388.
14.
Takebe T, Enomura M, Yoshizawa E, Kimura M, Koike H, Ueno Y, et al: Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell 2015;16:556-565.
15.
Takayama K, Nagamoto Y, Mimura N, Tashiro K, Sakurai F, Tachibana M, et al: Long-term self-renewal of human ES/iPS-derived hepatoblast-like cells on human laminin 111-coated dishes. Stem Cell Reports 2013;1:322-335.
16.
Mabuchi Y, Morikawa S, Suzuki S, Sunabori T, Okanoa H, Matsuzaki Y: Prospective isolation and identification of human mesenchymal stem cells by flow cytometry. Inflamm Regen 2009;29:73-78.
17.
Mabuchi Y, Houlihan DD, Akazawa C, Okano H, Matsuzaki Y: Prospective isolation of murine and human bone marrow mesenchymal stem cells based on surface markers. Stem Cells Int 2013;2013:507301.
18.
Basma H, Soto-Gutiérrez A, Yannam GR, Liu L, Ito R, Yamamoto T, et al: Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 2009;136:990-999.
19.
Duan Y, Ma X, Zou W, Wang C, Bahbahan IS, et al: Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells. Stem Cells 2010;28:674-686.
20.
Cai J, Zhao Y, Liu Y, Ye F, Song Z, Qin H, et al: Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 2007;45:1229-1239.
21.
Li Y, Chen J, Chen XG, Wang L, Gautam SC, Xu YX, et al: Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 2002;59:514-523.
22.
Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al: Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 2006;12:459-465.
23.
Tögel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C: Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol 2005;289:F31-F42.
24.
van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, Tilles AW, et al: Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology 2008;47:1634-1643.
25.
Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et al: Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013;499:481-484.
26.
Duncan AW, Dorrell C, Grompe M: Stem cells and liver regeneration. Gastroenterology 2009;137:466-481.
27.
Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, et al: Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol Hepatol 2009;24:70-77.
28.
Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al: Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS One 2007;2:e941.
29.
Meier RPH, Mahou R, Morel P, Meyer J, Montanari E, Muller YD, et al: Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice. J Hepatol 2015;62:634-641.
30.
Yang Y, Li J, Pan X, Zhou P, Yu X, Cao H, et al: Co-culture with mesenchymal stem cells enhances metabolic functions of liver cells in bioartificial liver system. Biotechnol Bioeng 2013;110:958-968.
31.
Togel F, Westenfelder C: The role of multipotent marrow stromal cells (MSCs) in tissue regeneration. Organogenesis 2011;7:96-100.
32.
Haynesworth SE, Baber MA, Caplan AI: Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol 1996;166:585-592.
33.
Dorshkind K: Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol 1990;8:111-137.
34.
Zhao D, Chen S, Duo S, Xiang C, Jia J, Guo M, et al: Promotion of the efficient metabolic maturation of human pluripotent stem cell-derived hepatocytes by correcting specification defects. Cell Res 2013;23:157-161.
35.
Tanaka M, Miyajima A: Oncostatin M, a multifunctional cytokine. Rev Physiol Biochem Pharmacol 2003;149:39-52.
36.
Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, et al: Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. EMBO J 1999;18:2127-2136.
37.
Snykers S, Vanhaecke T, Papeleu P, Luttun A, Jiang Y, Vander Heyden Y, et al: Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells. Toxicol Sci 2006;94:330-341.
38.
Lam SP, Luk JM, Man K, Ng KTP, Cheung CK, Rose-John S, et al: Activation of interleukin-6-induced glycoprotein 130/signal transducer and activator of transcription 3 pathway in mesenchymal stem cells enhances hepatic differentiation, proliferation, and liver regeneration. Liver Transpl 2010;16:1195-1206.
39.
Jung J, Zheng M, Goldfarb M, Zaret KS: Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 1999;284:1998-2003.
40.
Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, et al: Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 2009;19:1233-1242.
41.
Du Z, Wei C, Cheng K, Han B, Yan J, Zhang M, et al: Mesenchymal stem cell-conditioned medium reduces liver injury and enhances regeneration in reduced-size rat liver transplantation. J Surg Res 2013;183:907-915.
42.
Takayama K, Inamura M, Kawabata K, Katayama K, Higuchi M, Tashiro K, et al: Efficient generation of functional hepatocytes from human embryonic stem cells and induced pluripotent stem cells by HNF4α transduction. Mol Ther 2012;20:127-137.
43.
Berger DR, Ware BR, Davidson MD, Allsup SR, Khetani SR: Enhancing the functional maturity of induced pluripotent stem cell-derived human hepatocytes by controlled presentation of cell-cell interactions in vitro. Hepatology 2015;61:1370-1381.
44.
Takayama K, Kawabata K, Nagamoto Y, Kishimoto K, Tashiro K, Sakurai F, et al: 3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing. Biomaterials 2013;34:1781-1789.
45.
Kajiwara M, Aoi T, Okita K, Takahashi R, Inoue H, Takayama N, et al: Donor-dependent variations in hepatic differentiation from human induced pluripotent stem cells. Proc Natl Acad Sci USA 2012;109:12538-12543.
46.
Godoy P, Schmidt-Heck W, Natarajan K, Lucendo-Villarin B, Szkolnicka D, Asplund A, et al: Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells. J Hepatol 2015;63:934-942.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.