International Hepatology
Towards a bioengineered liver Dominique Franco⇑ Département Hospitalo-Universitaire Hepatinov; Hôpitaux Universitaires Paris-Sud; Univ Paris-Sud; Inserm, UMR972, France
COMENTARY ON: Vascularized and functional human liver from an iPSC-derived organ bud transplant. 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. Nature. 2013 Jul 25;499(7459):481–4. Copyright Ó (2013). Abstract reprinted by permission from Macmillan Publishers Ltd. Open access under CC BY-NC-ND license.
http://www.ncbi.nlm.nih.gov/pubmed/23823721 Abstract: A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liverspecific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. Open access under CC BY-NC-ND license. Despite agreement that portal injections of mature hepatocytes are partially effective in patients with hereditary metabolic liver
Keywords: Bioengineered liver; Stem cells; Liver differentiation. Received 22 August 2013; received in revised form 12 September 2013; accepted 12 September 2013 ⇑ Address: Hôpital Antoine-Béclère, 157 rue de la Porte-de-Trivaux, 92141 Clamart cedex, France. Tel.: +33 1 45 37 43 39. E-mail address:
[email protected].
diseases [1], the goal of replacing liver transplantation with this technique remains far off. Low functioning hepatocyte availability, a 50% cell loss during the procedure and difficulties in getting transplanted hepatocytes to integrate into liver plates and proliferate are the main causes of failure. The injection of stem cells rather than mature hepatocytes has given interesting results in specific rodent models [2], but there have been few trials in humans. Strategies for replacing liver transplantation have recently focused on the in vitro construction of bio-engineered livers. Decellularization-recellularization techniques have yielded liver-like transplantable organoids in small animals [3,4]. Much remains to be done, particularly concerning the cells used for repopulation, before this complex procedure can be applied to humans. Until recently, it was generally believed that liver organogenesis could not be reproduced in vitro, for the construction of a new liver de novo. In this study, Takabe and coworkers from Yokohama City University were able to construct a liver in a Petri dish, by culturing hepatic endoderm cells derived from human iPSCs (iPSC-HEs) with human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs). The cells were plated in two-dimensional conditions, but the human iPSCHEs self-organized into macroscopically visible, mechanically stable, manipulable, three-dimensional cell clusters — iPSCderived liver buds (iPSC-LBs) — four to eight days after seeding. In vitro, cells in human iPSC-LBs expressed early hepatic marker genes and were more functional than the hepatocyte-like cells generated from hiPSCs according to conventional procedures [5,6]. The FGF and BMP pathways, which play key roles in organogenesis, were upregulated when iPSC-HEs were co-cultured with HUVECs and MSCs. The entire process in the Petri dish closely resembled the development in vivo of liver buds from the foregut in human and mouse embryos. Human iPSC-LBs were transplanted into ectopic sites in immunodeficient mice, in which they grew and divided. Using the cranial window model, the authors observed the development of a human vasculature within the iPSC-LBs. Human blood vessels within the transplant became patent by connecting to the vessels of the mouse host at the edge of the transplant within 48 h of transplantation, resulting in a vascularized organoid. Two months later, the organoid had a vascular network, including sinusoids and hepatic cord organization with tight junctions. Most of the cells in the transplanted iPSC-LBs had developed into fully mature hepatocytes, with only a small percentage retaining fetal characteristics. Metabolic studies in mice with cranial windows or capsular iPSC-LB transplants, with drugs metabolized by different pathways in mice and humans, confirmed the appropriate functioning of transplanted human liver buds. Albumin production by
Journal of Hepatology 2014 vol. 60 j 455–456
International Hepatology Human mesenchymal cells (MSCs)
Co-culture
Liver specification
Human iPSCs
Human iPSC-HEs
4-6 d culture
3D liver bud
Transplantation in immunodeficient mice
Human endothelial cells (HUVECs)
Cranial window: ● study of vascularization ● histological studies
Kidney subcapsular space: ● functional studies
Mesentery in mice with acute liver failure: ● survival studies
Fig. 1. General scheme of the experiments.
hiPSC-LBs increased over time and was clearly stronger than that of human adult hepatocytes transplanted to the same sites. The functionality of a mesenteric transplant of hiPSC-LBs was further investigated in two models of acute liver failure in mice. In both models, hiPSC-LB transplantation significantly increased survival (Fig. 1). This constitutes a major breakthrough in the production of bioengineered livers. For the first time, a tiny, rudimentary liver has been generated from a few cells in a Petri dish, and the transplantation of this organoid ultimately protected mice against acute liver failure. The use of stromal cells to coax iPS-HEs into appropriate differentiation appears to be the key to obtaining the correct three-dimensional organization, maturation of cells and vascularization. Of course, much remains to be done to improve bud quality before this technique could be applied in clinical practice. For example, the ultrastructural organization of the bud does not entirely reflect the lobular organization of a normal liver. There is also no development of an external bile tree, potentially resulting in the accumulation of bile acids, which might jeopardize the long-term functioning of the transplanted bud. It also remains unclear whether similar results could be obtained in larger animals. Above all, such buds cannot be transplanted orthotopically. The best indication might, therefore, be the treatment of hereditary metabolic liver diseases, in which the host liver can be left in place and a genetically corrected bud implanted in the mesentery [7]. Buds could also be used as a bridging technique in patients with cirrhosis on the waiting list for transplantation, but bud generation may take too long for use in patients with acute liver failure. In addition to potential clinical applications, this study provides important information about the types of cells most suitable for liver construction, which could be applied to the recellularization of scaffolds. This technique may also prove a formidable tool for pharmacological studies. It is just
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one of many new technologies emerging in medical sciences. A mixture of these techniques, undoubtedly including layer-bylayer 3D printing [8], will probably ultimately be required for the de novo bioengineering of livers. Conflict of interest The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Dhawan A, Strom SC, Sokal E, Fox IJ. Human hepatocyte transplantation. Methods Mol Biol 2010;640:525–534. [2] Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology 2012;55:1193–1203. [3] Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 2010;16:814–820. [4] Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology 2011;53:604–617. [5] 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. [6] Touboul T, Hannan NR, Corbineau S, Martinez A, Martinet C, Branchereau S, et al. Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology 2010;51:1754–1765. [7] Dianat N, Steichen C, Vallier L, Weber A, Dubart-Kupperschmitt A. Human pluripotent stem cells for modelling human liver diseases and cell therapy. Curr Gene Ther 2013;13:120–132. [8] Xu T, Zhao W, Zhu JM, Albanna MZ, Yoo JJ, Atala A. Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology. Biomaterials 2013;34:130–139.
Journal of Hepatology 2014 vol. 60 j 455–456
