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  • br D culture systems and liver organoids As highlight by


    3D culture systems and liver organoids As highlight by the magazine The Economist in “The year of the organoid-The World in 2016”, another recent advance of the stem cell technology is the possibility to generate organoids. Organoids are based on the three-dimensional culture systems of stem cells from several different sources (iPSC, embryonic stem cells, adult stem cells and tissue-derived stem cells). The use of a 3D system offers the advantage to better preserve the cell-to cell contacts that allow cells to directly interact with each other and self-aggregate to reproduce the cellular organization of the organ. The majority of these methods are based on the use of matrigel matrix, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma [65] that is the major hint to induce maturation. In past years, several protocols have been developed for the generation of organoids cultures from iPSC-derived liver cells and from adult liver tissue. Two separate studies have used 3D culture conditions and matrigel to differentiate cholangiocytes derived from iPSC. Both studies were able to recapitulate the in vivo organization of cholangiocytes, as a single epithelial layer with a lumen and the expression of biliary specific markers [33] [44]. The culture in 3D conditions was improving cholangiocytes maturation but, in both cases, the end-point 3D structures were not propagated in culture. Another interesting study from Song et al. showed a system to induce 3D cell Mocetinostat (MGCD0103, MG0103) of iPDC-derived hepatocytes and stromal cells just using a microwell platform without any kind of matrix. These aggregates are then encapsulated in alginate capsule and transplanted into the peritoneal cavity of immune competent mouse therefore inducing further maturation of the hepatocytes [66]. Liver organoids have also been generated from adult somatic tissue-resident stem cells. In this specific case, they are derived directly from the liver tissue of the patient and represent an ideal tool for disease modeling, to test the efficacy and toxicity of drug compounds and they could offer an autologous source of cells for transplantation [67]. Based on the previous methodology developed by Clevers group, for the generation of Lgr5+ intestinal organoids, Broutier et al. have developed a system to obtain liver organoids from EpCam+ ductal cells from normal or diseased human livers [67]. These cells, when grown in matrigel and in low attachment plate in the presence of EGF, HGF, FGF and RSPO1, organize in 3D organoids with biliary progenitor identity that can possibly be differentiated toward the hepatic lineage. Contrary to iPSC-derived organoids, the advantage of this system is that tissue organoids can be cultured for long term (>1 year) still maintaining their genetic stability, can be cryopreserved and easily recovered after thawing, simplifying the process of cell banking [67]. In another recent study liver organoids were generated from the extrahepatic portion of the biliary tree (ECOs) [68]. Cells isolated by brushing or scraping the bile duct luminal surface grow in matrigel with a similar cocktail of specific growth factors and generate organoid structures. Interestingly, ECOs have the capacity to repopulate tubular polyglycolic acid (PGA) scaffolds, and still maintain their functionality and marker expression, thus representing a bioengineered tissue resembling the biliary epithelium. When transplanted in vivo in a mouse, whose midportion of the common bile duct (CBD) is removed, these bioengineered ducts are able to achieve biliary reconstruction. The ability of ECOs to replace a damaged CBD is a promising result for future regenerative medicine applications to manage liver diseases [68]. An attempt to generate organoids that mimic the human liver bud with multiple cell types has been otherwise done by Takebe and colleagues [69]. In this approach, the starting cell population is represented by iPSC that are differentiated into hepatic endoderm in a 2D monolayer. Subsequently, hepatic cells are co-cultured with human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (MSC) on matrigel coated plates. Under these conditions, the cells self-assemble to generate 3D clusters defined as liver buds since they resemble the human liver bud stage during liver development. When transplanted ectopically in NOD/SCID mice, the liver buds connect with the host vessels and become vascularized, improving their maturation and showing the presence of human specific metabolites in the serum of the mice. Excitingly, when transplanted in the mesentery, they improve survival in a TK-NOG mouse model of gancyclovir-induced liver failure [69].