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.
Highlights
Regenerative Medicine is an interdisciplinary field of research and clinical applications, focused on repair, replacement, or regeneration of cells, tissues, or organs to restore impaired function resulting from congenital defects, disease and trauma (Heidary Rouchi and Mahdavi-Mazdeh, 2015)
Cell Culture human pluripotent stem cells (hPSCs) hiPSCs were generated from human fibroblasts as previously described (Kyrkou et al, 2016), and the H1 hESC line was purchased from Wicell Research Institute (Madison, WI, United States). hPSCs were cultured on six-well tissue culture plates coated with hESC-qualified Matrigel (Corning, 354277) in mTeSR1 medium (StemCell Technologies, 05850) at 37◦C and 5% CO2
Since high serum is known to downregulate the expression of contractile proteins (Wanjare et al, 2013a), the key phenotypic feature of SMCs, we modified our differentiation strategy and developed a simple and quick differentiation protocol to induce the differentiation of hPSCs to contractile smooth muscle cells (cSMCs) using contractile differentiation medium (CDM) containing 2.5% fetal calf serum (FCS)
Summary
Regenerative Medicine is an interdisciplinary field of research and clinical applications, focused on repair, replacement, or regeneration of cells, tissues, or organs to restore impaired function resulting from congenital defects, disease and trauma (Heidary Rouchi and Mahdavi-Mazdeh, 2015). Vascularization remains a critical obstacle in engineering thicker, metabolically demanding organs, such as heart muscle, brain and liver as regenerating tissue over 100–200 μm exceeds the capacity of nutrient supply and waste removal by diffusion, and requires a vascular network (Carmeliet and Jain, 2000; Jain, 2005). It takes several weeks for a scaffold to become fully vascularized in vivo (Nillesen et al, 2007), and without a rapid and high level of vascularization of the transplanted grafts, the majority of cells fail to survive the early post-transplantation phase.
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