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

Abstract

Background The 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. Methods The 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. Results We 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. Conclusion Our 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.

Using Liver Organoids as Models to Study the Pathobiology of Rare Liver Diseases

“…Organized functional bile canaliculi system [70] In vitro expansion via modulation of Notch signaling iPSCs Hepatic endoderm, HscLCs, and endothelial cells…”

Hepatic organoids move from adolescence to maturity

“…A second approach uses separate pools of iPSCs, which were exposed to different culture conditions to generate different cell types (hepatic endoderm, endothelium or mesenchymal cells), and these cells are subsequently combined to produce hepatic organoids. This ‘ split pool’ approach was used to produce ‘limb buds’ with liver‐like functionality 44 and hepatic organoids with a microvasculature 45 . Vascularization is critically important because it allows hepatic organoids to grow to a larger size that is more representative of the liver environment.…”

“…This 'split pool' approach was used to produce 'limb buds' with liver-like functionality 44 and hepatic organoids with a microvasculature. 45 Vascularization is critically important because it allows hepatic organoids to grow to a larger size that is more representative of the liver environment. Cells in the interior of non-vascularized hepatic organoids undergo F I G U R E 3 A diagram of the high content screening (HCS) platform for analysis of microHOs.…”

Signaling pathways of liver regeneration: Biological mechanisms and implications

“… 79 In recent years, this pathway is demonstrated in a number of clinical observations and basic studies to exert an important effect on metabolism, vascular physiology, bile duct differentiation, and LR. 80 , 81 , 82 , 83 , 84 , 85 Moreover, great attention has been paid to LR. Notch pathway represents an earliest pathway that is activated within 15–30 min after PHx.…”