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.
Liver organoids have gained much attention in recent years for their potential applications to liver disease modeling and pharmacologic drug screening. Liver organoids produced in vitro reflect some aspects of the in vivo physiological and pathological conditions of the liver. However, the generation of liver organoids with perfusable luminal vasculature remains a major challenge, hindering precise and effective modeling of liver diseases. Furthermore, vascularization is required for large organoids or assembloids to closely mimic the complexity of tissue architecture without cell death in the core region. A few studies have successfully generated liver organoids with endothelial cell networks, but most of these vascular networks produced luminal structures after being transplanted into tissues of host animals. Therefore, formation of luminal vasculature is an unmet need to overcome the limitation of liver organoids as an in vitro model investigating different acute and chronic liver diseases. Here, we provide an overview of the unique features of hepatic vasculature under pathophysiological conditions and summarize the biochemical and biophysical cues that drive vasculogenesis and angiogenesis in vitro. We also highlight recent progress in generating vascularized liver organoids in vitro and discuss potential strategies that may enable the generation of perfusable luminal vasculature in liver organoids.
The liver is involved in physiological activities critical for survival. Chronic liver disease (CLD) frequently progresses to life-threatening liver failure, as evidenced by CLD patients’ high morbidity and mortality rates. Over the years, non-alcoholic fatty liver disease (NAFLD) has received significant attention as an etiology of CLD given its increasing prevalence and progression towards severe pathological conditions such as fibrosis. To answer the urgent need for effective therapeutics that treat CLD, an advanced cellular model, the liver organoid, is used to model and study the complex pathophysiology of NAFLD. Liver organoids recapitulate in vivo aspects of liver tissue such as 3-dimensional cell-cell and cell-extracellular matrix interactions and biomolecular gradients. Moreover, liver organoids can be readily generated from patient-specific liver tissues and induced pluripotent stem cells, enabling their use in a wide range of personalized clinical applications. In recent research, numerous attempts have been made to establish multicellular liver organoids capable of modeling disease phenotypes that involve parenchymal and non-parenchymal liver cells. In this review, we focus on recent advances in liver organoids and highlight the applicability of organoids for modeling NAFLD within the context of cellular sources and composition.
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