Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling

Stem Cell Research & Therapy

Research

Sang Kyum Kim¹  Yu Jin Jang²  Han-Jin Park³  Hyemin Kim³  Dong-Hun Woo⁴  Choongseong Han⁴  Kyun Yoo Chi⁵  Hyo Jin Kim⁵  Youngseok Lee⁵  Jihun Lee⁵  Gyeongmin Kim⁵  Jong-Hoon Kim⁵  Seongyea Jo⁶ 

[1]College of Pharmacy, Chungnam National University, 34134, Daejeon, South Korea

[2]Department of Molecular Biosciences, The University of Texas at Austin, 78712, Austin, TX, USA

[3]Department of Predictive Toxicology, Korea Institute of Toxicology, 34114, Daejeon, South Korea

[4]Department of Stem Cell Biology, NEXEL Co., Ltd, 07802, Seoul, South Korea

[5]Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, 02841, Seoul, South Korea

[6]Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, 02841, Seoul, South Korea

[7]Department of Predictive Toxicology, Korea Institute of Toxicology, 34114, Daejeon, South Korea

关键词: Liver organoid;    Vasculature;    Bile duct;    Notch;    Hepatic stellate cell;    Fibrosis;   

DOI  :  10.1186/s13287-023-03235-5

 received in 2022-08-04, accepted in 2023-01-03,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundThe generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves.MethodsThe mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines.ResultsWe found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage.ConclusionOur results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.

【 授权许可】

CC BY   
© The Author(s) 2023

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