Hepatic organoids (HOs) have been generated from human induced PSCs (iPSCs) and adult liver stem cells. However, organoids generated in previous studies do not harbor structural features of the liver such as the bile canaliculi network. In these previously reported liver organoids, disease modeling is limited to functional aspects of the different hepatic cell types, and they have not yet achieved the full potential of multicellular organoids in modeling systemic response. Further, most of these reported approaches are low throughput and generate pools of heterogeneous organoids, thus limiting their potential uses.
This article reports a stepwise, chemically defined, high throughput approach that enables the generation of a single Hepatic organoid (HOs) in each well of a 96-well plate. The presented approach generates organoids that consist of the 2 main liver cell types, hepatocytes and cholangiocytes, structurally arranged to recapitulate a functional bile canaliculi network. Using this unique structural organization of the HOs, the authors showed the first in vitro modeling of drug-induced cholestasis using troglitazone. Further, they showed that the organoids generated can be used to model the loss of bile canaliculi network16 and ductular reaction,17,18 which is closely associated with nonalcoholic fatty liver disease (NAFLD) progression.
H1 human embryonic stem cells were cultured and induced pluripotent stem cells with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live.
Figure 1: Stepwise generation of HOs from PSCs. (A) Schematic overview of HO generation from PSCs in a chemically defineand serum-free environment. This process involves the stepwise differentiation of PSCs into definitive endoderm (DE) and PFGunder 2-dimensional culture conditions. The PFG is further differentiated into HESs and, subsequently, into HEPSs in 3D,embedded in Matrigel. The HEPS is differentiated into organoids by using a 2-step approach in 3D suspension culture.Pathways targeted by growth factors, cytokines, and small molecules in each step of differentiation are highlighted. Shownalso are lineage-specific markers that reflect each cell-fate transition, as cells progressively commit to becoming hepatocytesand cholangiocytes in HO after approximately 50 days of culture. (B) Brightfield images of PSC, DE, and PFG and geneexpression analysis of pluripotency (SOX2, OCT4), endoderm (CXCR4, CER1), and PFG (OTX2, PDX1, CDX2, HNF4A) markerswith quantitative polymerase chain reaction. Relative expression was obtained by normalization to the PSC controls. Mean ±standard deviation of 3 independent experiments. (C) Immunostaining of PSC, DE, and PFG for anterior foregut marker OTX2and hepatic endoderm markers HNF4A and FOXA2. (D) Brightfield images and immunostaining of hepatobiliary markersSOX17, TBX3, HNF4A, and SOX9 in HES cultured in media conditions, containing all factors (A830-1, DEXA, CHIR99021, JAG-1, EGF, HGF), mitogen inducers (EGF and HGF), biliary inducers (JAG-1), hepatic inducers (A830-1, DEXA, CHIR99021), andnone (basal media) for 8 days. (E) Brightfield images of a single HO at days 27, 36, and 50 (left) and 48 individual organoids atday 50 of differentiation (right). (F) Immunostaining of HO with hepatocyte-specific (ALB and DPPIV) and cholangiocytespecific(CK7) markers at day 50 of differentiation. DAPI, 40,6-diamidino-2-phenylindole.
Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid–enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions.
The authors developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH. This organoids system can be used to study development of liver diseases and the effects of drugs. Further studies with these organoids are required to determine whether they are accurate models of human
liver diseases
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1Stem Cell and Regenerative Biology, Genome Institute of Singapore, Singapore. 2Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York City, New York. 3Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 4Institute of Medical Biology, A*STAR, Singapore. 5Skin Research Institute of Singapore, A*STAR, Singapore. 6Department of Surgery, University Surgical Cluster, National University Hospital, Singapore. 7Division of Gastroenterology and Hepatology, University Medicine Cluster,National University Hospital, Singapore. 8Department of Biochemistry, National University of Singapore, Singapore. 9Department of Biological Sciences, National University of Singapore. 10School of Biological Sciences, Nanyang Technological University, Singapore.
