Tissue Engineering Using Vascular Organoids From Human Pluripotent Stem Cell Derived Mural Cell Phenotypes

Abstract

Diffusion is a limiting factor in regenerating large tissues (100-200 µm) due to reduced nutrient supply and waste removal leading to low viability of the regenerating cells as neovascularization of the implant by the host is a slow process. Thus, generating prevascularized tissue engineered constructs, in which endothelial (ECs) and mural (MCs) cells, such as smooth muscle cells (SMCs), and pericytes (PCs), are preassembled into functional in vitro vessels capable of rapidly connecting to the host vasculature could overcome this obstacle. Toward this purpose, using feeder-free and low serum conditions, we developed a simple, efficient and rapid in vitro approach to induce the differentiation of human pluripotent stem cells-hPSCs (human embryonic stem cells and human induced pluripotent stem cells) to defined SMC populations (contractile and synthetic hPSC-SMCs) by extensively characterizing the cellular phenotype (expression of CD44, CD73, CD105, NG2, PDGFRβ, and contractile proteins) and function of hPSC-SMCs. The latter were phenotypically and functionally stable for at least 8 passages, and could stabilize vessel formation and inhibit vessel network regression, when co-cultured with ECs in vitro. Subsequently, using a methylcellulose-based hydrogel system, we generated spheroids consisting of EC/hPSC-SMC (vascular organoids), which were extensively phenotypically characterized. Moreover, the vascular organoids served as focal starting points for the sprouting of capillary-like structures in vitro, whereas their delivery in vivo led to rapid generation of a complex functional vascular network. Finally, we investigated the vascularization potential of these vascular organoids, when embedded in hydrogels composed of defined extracellular components (collagen/fibrinogen/fibronectin) that can be used as scaffolds in tissue engineering applications. In summary, we developed a robust method for the generation of defined SMC phenotypes from hPSCs. Fabrication of vascularized tissue constructs using hPSC-SMC/EC vascular organoids embedded in chemically defined matrices is a significant step forward in tissue engineering and regenerative medicine.

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity

“…A simple method to generate vascular spheroids is to aggregate vascular cells, either stem cell-derived or primary, directly into 3D spheres. This can be done using the hanging-drop technique ( Markou et al, 2020 ) or using an ultra—low attachment culture substrate ( Moldovan et al, 2017 ). Interestingly, distinct cell types mixed together to form spheroids tend to spontaneously organize themselves into a structural hierarchy reminiscent of a native vasculature.…”

“…Interestingly, distinct cell types mixed together to form spheroids tend to spontaneously organize themselves into a structural hierarchy reminiscent of a native vasculature. For instance, Markou et al generated small vascular spheroids (∼100 μm) from iPSC-ECs and iPSC-SMCs ( Markou et al, 2020 ). The iPSC-ECs spontaneously formed the outer lining of these spheroids, allowing them to be in direct contact with the culture medium, whereas the iPSC-SMCs lined beneath the iPSC-ECs, forming the interior layer ( Markou et al, 2020 ).…”

“…For instance, Markou et al generated small vascular spheroids (∼100 μm) from iPSC-ECs and iPSC-SMCs ( Markou et al, 2020 ). The iPSC-ECs spontaneously formed the outer lining of these spheroids, allowing them to be in direct contact with the culture medium, whereas the iPSC-SMCs lined beneath the iPSC-ECs, forming the interior layer ( Markou et al, 2020 ). Despite being a relatively crude model, these spheroids emulate some aspects of vascular anatomy that are not present in a 2D culture.…”

Induced Pluripotent Stem Cells (iPSCs) in Vascular Research: from Two- to Three-Dimensional Organoids

“…The application possibilities of the organoids are numerous, starting from investigating high blood pressure and shear stress effects on ECs, to the effects of inflammatory factors on SMC behaviour and phenotype switching, or cross-talk between ECs and VSMCs during stress. Furthermore, transplantation of pre-vascularized tissues could accelerate wound healing by promoting the connection of the transplant with the surrounding host tissue [ 58 ]. In future, it would be advantageous to adapt the vascular organoids to contain VSMCs beneath the already established cell types of the vascular system ECs and PCs to better resemble the physiological environment.…”

Current State of the Art in Ventricle Tissue Engineering

“…The field of cardiac tissue engineering includes heart muscle tissue (2, 3), aortic valves (5), vascular grafts (6)(7)(8), vascular networks (9,10) and Purkinje networks, left ventricles (11), and whole hearts (12) (Figure 1). While heart muscle tissue engineering is now an established field (3), the same is not true for ventricle bioengineering, a niche field and the focus on this review article.…”