Dataset Information

Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids.

ABSTRACT: The induction of tissue-specific vessels in in vitro living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b⁺ putative liver sinusoidal progenitors (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate and sustain hepatocyte-like cells neighbored by divergent endothelial subsets composed of CD32b^lowCD31^high, LYVE1⁺STAB1⁺CD32b^highCD31^lowTHBD^-vWF^-, and LYVE1^-THBD⁺vWF⁺ cells. Wnt2 mediated sinusoidal-to-hepatic intercellular crosstalk potentiates hepatocyte differentiation and branched endothelial network formation. Intravital imaging revealed iLSEP developed fully patent human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enabled correction of in vitro clotting time with Factor V, VIII, IX, and XI deficient patients' plasma and rescued the severe bleeding phenotype in hemophilia A mice upon transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.

SUBMITTER: Saiki N

PROVIDER: S-EPMC11245015 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids.

Saiki Norikazu N Nio Yasunori Y Yoneyama Yosuke Y Kawamura Shuntaro S Iwasawa Kentaro K Kawakami Eri E Araki Kohei K Fukumura Junko J Sakairi Tsuyoshi T Kono Tamaki T Ohmura Rio R Koido Masaru M Funata Masaaki M Thompson Wendy L WL Cruz-Encarnacion Pamela P Chen Ya-Wen YW Takebe Takanori T

bioRxiv : the preprint server for biology 20240704

The induction of tissue-specific vessels in <i>in vitro</i> living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b<sup>+</sup> putative liver sinusoidal progenitors (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate and sustain hepatocyte-like cells neighbored ...[more]

PMID: 39005378

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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

Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids.

Publications

Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids.

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