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

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.

In Vitro Generation of Luminal Vasculature in Liver Organoids: From Basic Vascular Biology to Vascularized Hepatic Organoids.

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.

Generation of cytochrome P450 enzyme-polymorphic human induced pluripotent stem cell lines with CYP2D6*10/CYP3A5*3C and CYP2D6*10/CYP2C19*2.

Cytochrome P450 (CYP) reaction phenotyping has become crucial for predicting drug reactions and side effects. Single nucleotide polymorphisms (SNPs) in CYP genes alter drug metabolism capacity and cause unexpected drug-related reactions. Here, we established two human induced pluripotent stem cell (hiPSC) lines with pharmacologically important SNPs in CYP2D6 in conjunction with CYP2C19 or CYP3A5 genes. These hiPSC lines can serve as valuable resources for expanding our understanding of the relationships between genotypes and drug reactions.

Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs.

The bleeding disorder hemophilia A (HA) is caused by a single-gene (F8) defect and its clinical symptom can be substantially improved by a small increase in the plasma coagulation factor VIII (FVIII) level. In this study, we used F8-defective human induced pluripotent stem cells from an HA patient (F8d-HA hiPSCs) and F8-corrected (F8c) HA hiPSCs produced by CRISPR/Cas9 genome engineering of F8d-HA hiPSCs. We obtained a highly enriched population of CD157+ cells from CRISPR/Cas9-edited F8c-HA hiPSCs. These cells exhibited multiple cellular and functional phenotypes of endothelial cells (ECs) with significant levels of FVIII activity, which was not observed in F8d-HA hiPSC-ECs. After transplantation, the engineered F8c-HA hiPSC-ECs dramatically changed bleeding episodes in HA animals and restored plasma FVIII activity. Notably, grafting a high dose of ECs substantially reduced the bleeding time during multiple consecutive bleeding challenges in HA mice, demonstrating a robust hemostatic effect (90% survival). Furthermore, the engrafted ECs survived more than 3 months in HA mice and reversed bleeding phenotypes against lethal wounding challenges. We also produced F8c-HA hiPSC-derived 3D liver organoids by assembling three different cell types in microwell devices and confirmed its therapeutic effect in HA animals. Our data demonstrate that the combination of genome-engineering and iPSC technologies represents a novel modality that allows autologous cell-mediated gene therapy for treating HA.