Establishment of human induced pluripotent stem cell-derived hepatobiliary organoid with bile duct for pharmaceutical research use.

In vitro models of the human liver are promising alternatives to animal tests for drug development. Currently, primary human hepatocytes (PHHs) are preferred for pharmacokinetic and cytotoxicity tests. However, they are unable to recapitulate the flow of bile in hepatobiliary clearance owing to the lack of bile ducts, leading to the limitation of bile analysis. To address the issue, a liver organoid culture system that has a functional bile duct network is desired. In this study, we aimed to generate human iPSC-derived hepatobiliary organoids (hHBOs) consisting of hepatocytes and bile ducts. The two-step differentiation process under 2D and semi-3D culture conditions promoted the maturation of hHBOs on culture plates, in which hepatocyte clusters were covered with monolayered biliary tubes. We demonstrated that the hHBOs reproduced the flow of bile containing a fluorescent bile acid analog or medicinal drugs from hepatocytes into bile ducts via bile canaliculi. Furthermore, the hHBOs exhibited pathophysiological responses to troglitazone, such as cholestasis and cytotoxicity. Because the hHBOs can recapitulate the function of bile ducts in hepatobiliary clearance, they are suitable as a liver disease model and would be a novel in vitro platform system for pharmaceutical research use.

Wang L ，Koui Y ，Kanegae K ，Kido T ，Tamura-Nakano M ，Yabe S ，Tai K ，Nakajima Y ，Kusuhara H ，Sakai Y ，Miyajima A ，Okochi H ，Tanaka M ... -  《-》

Generation of hepatobiliary organoids from human induced pluripotent stem cells.

Human induced pluripotent stem cell (hiPSC)-derived liver modeling systems have the potential to overcome the shortage of donors for clinical application and become a model for drug development. Although several strategies are available to generate hepatic micro-tissues, few have succeeded in generating a liver organoid with hepatobiliary structure from hiPSCs. At differentiation stages I and II (day 1-15), 25% of mTeSR™ culture medium was added to hepatic differentiation medium to induce endodermal and mesodermal commitment and thereafter hepatic and biliary co-differentiation. At stage III (day 15-45), 10% cholesterol+ MIX was added to the maturation medium to promote the formation and maturation of the hepatobiliary organoids. Phenotypes and functions of organoids were determined by specific markers and multiple functional assays both in vitro and in vivo. In this system, hiPSCs were induced to form 3D hepatobiliary organoids and to some extent recapitulated key aspects of early hepatogenesis in a parallel fashion. The organoids displayed a series of functional attributes. Specifically, the induced hepatocyte-like cells could take up indocyanine green, accumulate lipid and glycogen, and displayed appropriate secretion ability (albumin and urea) and drug metabolic ability (CYP3A4 activity and inducibility); the biliary structures in the system showed gamma glutamyltransferase activity and the ability to efflux rhodamine and store bile acids. Furthermore, after transplantation into the immune-deficient mice, the organoids survived for more than 8 weeks. This is the first time that functional hepatobiliary organoids have been generated from hiPSCs. The organoid model will be useful for in vitro studies of the molecular mechanisms of liver development and has important potential in the therapy of liver diseases. Herein, we established a system to generate human induced pluripotent stem cell-derived functional hepatobiliary organoids in vitro, without any exogenous cells or genetic manipulation. To some extent this model was able to recapitulate several key aspects of hepatobiliary organogenesis in a parallel fashion, holding great promise for drug development and liver transplantation.

Wu F ，Wu D ，Ren Y ，Huang Y ，Feng B ，Zhao N ，Zhang T ，Chen X ，Chen S ，Xu A ... -  《-》

Generation of human hepatobiliary organoids with a functional bile duct from chemically induced liver progenitor cells.

Liver disease imposes a significant medical burden that persists due to a shortage of liver donors and an incomplete understanding of liver disease progression. Hepatobiliary organoids (HBOs) could provide an in vitro mini-organ model to increase the understanding of the liver and may benefit the development of regenerative medicine. In this study, we aimed to establish HBOs with bile duct (BD) structures and mature hepatocytes (MHs) using human chemically induced liver progenitor cells (hCLiPs). hCLiPs were induced in mature cryo-hepatocytes using a small-molecule cocktail of TGF-β inhibitor (A-83-01, A), GSK3 inhibitor (CHIR99021, C), and 10% FBS (FAC). HBOs were then formed by seeding hCLiPs into ultralow attachment plates and culturing them with a combination of small molecules of Rock-inhibitor (Y-27632) and AC (YAC). These HBOs exhibited bile canaliculi of MHs connected to BD structures, mimicking bile secretion and transportation functions of the liver. The organoids showed gene expression patterns consistent with both MHs and BD structures, and functional assays confirmed their ability to transport the bile analogs of rhodamine-123 and CLF. Functional patient-specific HBOs were also successfully created from hCLiPs sourced from cirrhotic liver tissues. This study demonstrated the potential of human HBOs as an efficient model for studying hepatobiliary diseases, drug discovery, and personalized medicine.

Li P ，Miyamoto D ，Fukumoto M ，Kawaguchi Y ，Yamashita M ，Tetsuo H ，Adachi T ，Hidaka M ，Hara T ，Soyama A ，Matsushima H ，Imamura H ，Kanetaka K ，Gu W ，Eguchi S ... -  《Stem Cell Research & Therapy》

Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids.

The development of hepatic models capable of long-term expansion with competent liver functionality is technically challenging in a personalized setting. Stem cell-based organoid technologies can provide an alternative source of patient-derived primary hepatocytes. However, self-renewing and functionally competent human pluripotent stem cell (PSC)-derived hepatic organoids have not been developed. We developed a novel method to efficiently and reproducibly generate functionally mature human hepatic organoids derived from PSCs, including human embryonic stem cells and induced PSCs. The maturity of the organoids was validated by a detailed transcriptome analysis and functional performance assays. The organoids were applied to screening platforms for the prediction of toxicity and the evaluation of drugs that target hepatic steatosis through real-time monitoring of cellular bioenergetics and high-content analyses. Our organoids were morphologically indistinguishable from adult liver tissue-derived epithelial organoids and exhibited self-renewal. With further maturation, their molecular features approximated those of liver tissue, although these features were lacking in 2D differentiated hepatocytes. Our organoids preserved mature liver properties, including serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. The organoids exhibited significant toxic responses to clinically relevant concentrations of drugs that had been withdrawn from the market due to hepatotoxicity and recapitulated human disease phenotypes such as hepatic steatosis. Our organoids exhibit self-renewal (expandable and further able to differentiate) while maintaining their mature hepatic characteristics over long-term culture. These organoids may provide a versatile and valuable platform for physiologically and pathologically relevant hepatic models in the context of personalized medicine. A functionally mature, human cell-based liver model exhibiting human responses in toxicity prediction and drug evaluation is urgently needed for pre-clinical drug development. Here, we develop a novel human pluripotent stem cell-derived hepatocyte-like liver organoid that is critically advanced in terms of its generation method, functional performance, and application technologies. Our organoids can contribute to the better understanding of liver development and regeneration, and provide insights for metabolic studies and disease modeling, as well as toxicity assessments and drug screening for personalized medicine.

Mun SJ ，Ryu JS ，Lee MO ，Son YS ，Oh SJ ，Cho HS ，Son MY ，Kim DS ，Kim SJ ，Yoo HJ ，Lee HJ ，Kim J ，Jung CR ，Chung KS ，Son MJ ... -  《-》

Efficient and Controlled Generation of 2D and 3D Bile Duct Tissue from Human Pluripotent Stem Cell-Derived Spheroids.

Tian L ，Deshmukh A ，Ye Z ，Jang YY ... -  《-》