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Generation of liver bipotential organoids with a small-molecule cocktail.

ABSTRACT: Understanding the mechanism of how cholangiocytes (liver ductal cells) are activated upon liver injury and specified to hepatocytes would permit liver regenerative medicine. Here we achieved long-term in vitro expansion of mouse liver organoids by modulating signaling pathways with a combination of three small-molecule compounds. CHIR-99021, blebbistatin, and forskolin together maintained the liver organoids in bipotential stage with both cholangiocyte- and hepatocyte-specific gene expression profiles and enhanced capacity for further hepatocyte differentiation. By employing a chemical approach, we demonstrated that Wnt/?-catenin, NMII-Rac, and PKA-ERK are core signaling pathways essential and sufficient for mouse liver progenitor expansion. Moreover, the advanced small-molecule culture of bipotential organoids facilitates the ex vivo investigation of liver cell fate determination and the application of organoids in liver regenerative medicine.

SUBMITTER: Wang X

PROVIDER: S-EPMC7683013 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Generation of liver bipotential organoids with a small-molecule cocktail.

Wang Xin X Ni Chao C Jiang Ning N Wei Jinsong J Liang Jianqing J Zhao Bing B Lin Xinhua X

Journal of molecular cell biology 20200801 8

Understanding the mechanism of how cholangiocytes (liver ductal cells) are activated upon liver injury and specified to hepatocytes would permit liver regenerative medicine. Here we achieved long-term in vitro expansion of mouse liver organoids by modulating signaling pathways with a combination of three small-molecule compounds. CHIR-99021, blebbistatin, and forskolin together maintained the liver organoids in bipotential stage with both cholangiocyte- and hepatocyte-specific gene expression pr ...[more]

PMID: 32232340

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Project description:BackgroundThe use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.MethodsThe peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.ResultsCD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

Project description:Introduction: The molecular regulation mechanism of fat deposition in bovine and its improvement on beef quality are important research directions in the livestock industry. The research of molecular mechanisms that govern the regulation and differentiation of adipocytes may conduct to understand the mechanism of obesity, lipid disorders, and fat deposition. In the recent decade, small-molecule compounds have been widely used in reprogramming and transdifferentiation fields, which can promote the induction efficiency, replace exogenous genes, or even induce cell fate conversion alone. Furthermore, small-molecule compound induction is expected to be a novel approach to generate new cell types from somatic cells in vitro and in vivo. Methods: In this study, we established rapid chemically induced platform for transdifferentiation of bovine ear fibroblasts into adipocyte-like cells using a small-molecule cocktail (Repsox, VPA, TTNPB). The chemically induced adipocytes (CiADCs) were characterized by lipid staining, qRT-PCR and WB. Bovine natural adipocytes were used as positive control, and the expression of adipocyte-related marker genes in CiADCs were analyzed. Moreover, RNA-Seq explore the mechanism of RVB in the regulation of Bovine adipocyte transdifferentiation. Results: In this study, the chemically induced adipocytes (CiADCs) could be identified as early as day 6. The CiADCs appeared to be circular and rich of lipid droplets. The adipocyte-specific genes of LPL, PPARγ, IGF1, GPD1, C/EBPδ, ADIPOQ, PCK2, FAS, C/EBPβ, PPARGC1A, C/EBPα, and CFD were detected to be significantly upregulated in both CiADCs and natural adipocytes. Western blot analysis also confirmed the increase C/EBPα and PPARγ protein level in induced adipocytes (CiADCs-6d) treated with RVB. In addition, we also found that the signaling pathways (PPAR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and ECM-receptor interaction) regulated by the DEGs played a vital role in adipogenesis. Discussion: In the present study, a combination of small-molecule compounds RVB was used to transdifferentiate bovine ear fibroblasts into the chemically-induced adipocyte cells (CiADCs) that have a large number of lipid droplets. Importantly, the small-molecule cocktail significantly shortened the reprogramming turnaround time. The morphology of CiADCs is close to the "ring type" of natural differentiated adipocytes on sixth day. And, the CiADCs showed similar adipocyte-specific gene expression patterns to natural adipocytes. Furthermore, RVB increased protein expression of PPARγ and C/EBPα in the chemically-induced adipocytes (CiADCs-6d). Our findings reveal that the signaling pathways of C/EBPα and PPARγ play pivotal roles in this transdifferentiation process. In addition, we also found that the signaling pathways (PPAR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, MAPK signaling pathway, and ECM-receptor interaction) regulated by the DEGs played a vital role in adipogenesis. In general, this study provides valuable evidence to deepen our understanding of the molecular mechanism of small molecule cocktails in regulating adipogenesis.

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Generation of liver bipotential organoids with a small-molecule cocktail.

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Generation of liver bipotential organoids with a small-molecule cocktail.

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