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

Abstract

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.

Keywords: Blood vessel; Liver; Organoid; Stem cell; Vascularization.

Fig. 1

Interactions between liver sinusoidal endothelial cells and other liver-resident cells in healthy (A) and pathological (B) conditions. LSEC: liver sinusoidal endothelial cell, qHSC: quiescent hepatic stellate cell, aHSC: activated hepatic stellate cell, NO: nitric oxide, eNOS: endothelial nitric oxide synthase, DAMP: damage-associated molecular pattern, ECM: extracellular matrix, HH: hedgehog, ICAM-1: intercellular adhesion molecule-1, VCAM-1: vascular cell adhesion molecule-1, VAP-1: vascular adhesion protein-1, IL-6: interleukin-6, TNFα: tumor necrosis factor alpha, MCP-1: monocyte chemoattractant protein-1.

Fig. 2

Schematic illustration of angiogenesis and related signaling path-ways. (A) The angiogenesis process. (B) Signaling pathways in tip cell/stalk cell specification. EC: endothelial cell, BM: basement membrane, VE-cadherin: vascular endothelial cadherin, VEGF: vascular endothelial growth factor, VEGFR: VEGF receptor, DLL: Delta-like ligand, NICD: Notch intracellular domain.

Fig. 3

Current approaches used for generating vascularized liver organoids. (A) Self-organized liver organoids with vasculature by assembling different cell types derived from multiple cell sources. (B) Two different approaches used for printing liver tissue-like architectures from hepatocytes, fibroblast, and ECs. (C) Illustrations representing the strategies for generating microfluidic device-based liver organoids with perfusable vessels. iPSC: induced pluripotent stem cell, iPSC-HE: iPSC-derived hepatic endoderm, iPSC-EC: iPSC-derived endothelial cell, HUVEC: human umbilical vein endothelial cell, LSEC: liver sinusoidal endothelial cell, MSC: mesenchymal stem cell, HSC: hepatic stellate cell, iPSC-STM: iPSC-derived septum transversum mesenchyme, ECM: extracellular matrix, ULA: ultra-low attachment, GATA6: GATA binding transcription factor 6, PROX1: Prospero homeobox 1, ATF5: activating transcription factor 5, CYP3A4: cytochrome P450 3A4, PHH: primary human hepatocyte, 3D: three dimentional, KC: Kupffer cell, Mф: macrophage.