Hyo Jin Kim, Gyeongmin Kim, Kyun Yoo Chi, Hyemin Kim, Yu Jin Jang, Seongyea Jo, Jihun Lee, Youngseok Lee, Dong-Hun Woo, Choongseong Han, Sang Kyum Kim, Han-Jin Park & Jong-Hoon Kim
Stem Cell Res Ther 14, 19 (2023). https://doi.org/10.1186/s13287-023-03235-5
Copyright © Authors 2023
This article is licensed under a Creative Commons Attribution 4.0 International License (CC BY).
Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling
This study demonstrates that these multilineage liver organoids, assembled using PrimeSurface ultra-low attachement plates, can effectively mimic interactions between different liver cell types, advancing the potential for precise in vitro modeling of liver diseases. These plates are designed to prevent cell adhesion, which helps promote the formation of 3D structures like organoids.
Key Highlights:
- Organoid Formation: The researchers used PrimeSurface plates to culture hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells together, allowing them to self-organize into complex, functional liver organoids.
This technique ensured that the organoids developed proper vascular networks and bile ducts, making them more physiologically relevant for disease modeling and drug testing.
Role of HscLCs: Incorporating HscLCs was crucial for forming functional microvasculature within the organoids. Their presence reduced cell death and promoted organoid growth.
Notch Signaling: Precise modulation of Notch signaling during differentiation was essential for developing both bile ducts and vasculature in the organoids.
Structural Validation: Advanced imaging techniques confirmed that the organoids contained well-distributed, perfusable blood vessels.
Fibrosis Modeling: When exposed to pro-fibrotic cytokines, the organoids exhibited early signs of fibrosis, including upregulation of specific genes and structural changes, indicating their potential for disease modeling
Background
The liver is a complex organ composed of hepatocytes and other cells. Hepatocytes (parenchymal cells) perform the majority of functions, but non-parenchymal cells such as hepatic astrocytes and Kupffer cells also play an important role. In vitro modeling of liver disease is important to reproduce the interaction between parenchymal and non-parenchymal cells. Recently, various types of multilineage liver organoids (mLOs) can be generated from human iPS cells, however, the assembly and concurrent differentiation of multiple cell types in individual mLOs remain to be a major challenge. While, most studies have focused on the vascularization of mLOs in host tissue after transplantation in vivo, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves.
Research Achievements
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 PrimeSurface® 96U plate. The results from this study demonstrated that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggested that their application can advance the precise modeling of liver diseases in vitro.
Use of PrimeSurface in this Study
- PrimeSurface® 96U plate was used to generate mLOs:
- Cells：To optimize the ratio and the composition of cell types in mLOs (total of 5 × 104 cells), different ratios of HE/EC/HscLC and combinations (HE + EC + HscLC; HE + HscLC; HE only) of cell types were assembled and seeded.
- Medium: cold multilineage liver organoid differentiation medium (DM)
- Culture: days 1 and 3, cold DM was added to the organoid culture. Five days after aggregation, mLOs were transferred to ultra-low attachment 24-well plates and further cultured in DM. The medium was replaced every 3 days.
Results: presumptive sprouting of EC networks at the periphery of organoids with HscLCs, while a decrease in organoid size in mLO w/o HscLCs.
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