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Generation of human pluripotent stem cell-derived fused organoids with oligodendroglia and myelin.

ABSTRACT: Current knowledge on brain oligodendrogenesis, composed of three sequential waves from different regions, is mainly obtained from rodent studies. Oligodendroglial development and myelination in the rodent brain may not fully mirror those processes in the human brain. Here, we provide a step-by-step protocol for generating fused forebrain organoids derived from human pluripotent stem cells. These fused organoids recapitulate human oligodendroglial developments, offering innovative insights into human myelination research in vitro. For complete details on the use and execution of this protocol, please refer to Cameron-Curry and Le Douarin (1995), Kessaris et al. (2006), and Kim et al. (2019).

SUBMITTER: Kim H

PROVIDER: S-EPMC8038946 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Generation of human pluripotent stem cell-derived fused organoids with oligodendroglia and myelin.

Kim Hyosung H Jiang Peng P

STAR protocols 20210407 2

Current knowledge on brain oligodendrogenesis, composed of three sequential waves from different regions, is mainly obtained from rodent studies. Oligodendroglial development and myelination in the rodent brain may not fully mirror those processes in the human brain. Here, we provide a step-by-step protocol for generating fused forebrain organoids derived from human pluripotent stem cells. These fused organoids recapitulate human oligodendroglial developments, offering innovative insights into h ...[more]

PMID: 33851141

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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.

Dataset Information

Generation of human pluripotent stem cell-derived fused organoids with oligodendroglia and myelin.

Publications

Generation of human pluripotent stem cell-derived fused organoids with oligodendroglia and myelin.

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