Updated perspectives on vascular cell specification and pluripotent stem cell-derived vascular organoids for studying vasculopathies

Abstract

Vasculopathy is a pathological process occurring in the blood vessel wall, which could affect the haemostasis and physiological functions of all the vital tissues/organs and is one of the main underlying causes for a variety of human diseases including cardiovascular diseases. Current pharmacological interventions aiming to either delay or stop progression of vasculopathies are suboptimal, thus searching novel, targeted, risk-reducing therapeutic agents, or vascular grafts with full regenerative potential for patients with vascular abnormalities are urgently needed. Since first reported, pluripotent stem cells (PSCs), particularly human-induced PSCs, have open new avenue in all research disciplines including cardiovascular regenerative medicine and disease remodelling. Assisting with recent technological breakthroughs in tissue engineering, in vitro construction of tissue organoid made a tremendous stride in the past decade. In this review, we provide an update of the main signal pathways involved in vascular cell differentiation from human PSCs and an extensive overview of PSC-derived tissue organoids, highlighting the most recent discoveries in the field of blood vessel organoids as well as vascularization of other complex tissue organoids, with the aim of discussing the key cellular and molecular players in generating vascular organoids.

Keywords: Blood vessel; Embryonic stem cell; Induced pluripotent stem cell; Organoid vascularization; Pluripotent stem cell; Tissue-engineered vascular graft; Vascular disease; Vascular endothelial cell; Vascular organoid; Vascular smooth muscle cell; Vasculopathy.

Graphical Abstract

Figure 1

Spatial arrangement of vascular system. Endothelial cells constitute the inner layer of all types of blood vessels, where smooth muscle cells make up the middle layer (except for capillaries). Moreover, the middle layer of veins is thinner than that of arteries. Arterioles and venules are the secondary vessels of arteries and veins, respectively. As for capillaries, pericytes can be readily found just in-between the endothelial cells and basement membrane.

Figure 2

Schematic diagram illustrating the following induction processes: (1) iPSCs can be differentiated into neural crest cells by FGF-2, followed by further differentiation on stiff matrix, with serum-free medium containing TGF-β1; differentiated VSMCs were then seeded onto biodegradable scaffold, followed by cyclic stretching to stimulate cell maturation and collagen synthesis; (2) iPSCs were differentiated into mesodermal cells using BMP4 combined with glycogen synthase kinase 3 inhibitor (CHIR99021 or CP21), and then specified into two different fates, VSMCs and VECs, with either PDGF − BB + Activin-A or VEGF + Forskolin; (3) Parallel-plate flow chamber can maintain a consistent shear stress, which alters the cytoskeleton of iPSC and enhanced their differentiation capacities to VECs. BMP4, bone morphogenetic protein 4; FGF-2, basic fibroblast growth factor; iPSCs, induced pluripotent stem cells; PDGF-BB, platelet-derived growth factor-BB; TGF-β1, transforming growth factor beta 1; VECs, vascular endothelial cells; VSMCs, vascular smooth muscle cells.

Figure 3

Strategies to manufacture tissue-engineered vascular graft (TEVG). (A) Co-culture of VECs and hASCs on de-cellularized small intestinal submucosa: VECs from five different sources (HUVECs, hCECs, hPAECs, iCell-ECs hiPSC-ECs) were co-cultured with hASCs under controlled stimuli (growth factors, 3D hydrogel) in bio-incubator, resulting in spontaneous formation of vascular network via self-organization. Importantly, VECs were surrounded by hASCs as mural cells, forming small calibre lumens within the 3D hydrogel. (B) iPSC-derived VSMCs and VECs: Somatic cells can be isolated either from patients themselves or from other healthy individuals and then reprogrammed into hiPSCs. For homologous hiPSCs, gene-editing tool (CRISPR-Cas9) enables acquisition of hypo-immunogenic hiPSCs (B2m^−/−/Ciita^−/−/Cd47). Afterwards, hiPSC-VSMCs were seeded onto a scaffold with appropriate stiffness and then transferred into bio-incubator, where proper temperature, mechanical stimulation, humidity, pH, and growth factors are controlled precisely to generate preliminary TEVG. Thereafter, hiPSC-VECs coated the lumen of preliminary TEVG to create the final TEVG which are ready for vascular grafting. EC, endothelial cells; hASCs, human adipose tissue-derived stromal cells; hCEC, human cardiac endothelial cell; hPAEC, human pulmonary artery endothelial cell; HUVECs, human umbilical vein endothelial cells; iCell-ECs, iPSC-derived endothelial cells from Cellular Dynamics International; VECs, vascular endothelial cells; VSMCs, vascular smooth muscle cells.

Figure 4

Schematic representation of in vitro generation of hPSC-derived vascular organoids. Using this multi-step protocol as reported by Wimmer et al. vascular organoids can be successfully created from hPSCs (human pluripotent stem cells) and maintained in culture dishes, which could be used for pathophysiological study, disease modelling, high-throughput drug screening, and in vivo transplantation, respectively.

Figure 5

A diagrammatic representation of the original and vascularized whole-brain organoid protocols reported by (A) Lancaster and Knoblich as well as recently developed strategies of vascularization of human whole-brain organoids by (B) Pham et al., (C) Wörsdörfer et al., (D) Cakir et al., and (E) Ham et al.¹⁵⁶).

Figure 6

Applications of in vitro vascular organotypic technology. (A) Pluripotent stem cell-derived vascular organoids may provide new insights into pivotal developmental events or signalling pathways during organogenesis. (B) Patient-derived or gene-modified vascular organoids can be employed to study disease development, progression, and oncogenesis. (C) Patient-compatible vascular organoids for application in regenerative medicine. (D) Scalable, translatable vascular organoid-based platforms for high-throughput drug screening and development.