"INTRODUCTION: Regenerating large tissues requires an intimate supply of the host vascular network, a slow process leading to low viability of the regenerating cells. A solution to this obstacle is the generation of prevascularized tissue engineered constructs. Since Endothelial (ECs) and Mural (MCs) cells, such as smooth muscle cells (SMCs), and pericytes (PCs), are the cellular components of blood vessels and their interactions are crucial for neovascularization, both cell types and their arrangement into correct spatial organization are required in order to rescue tissue engineered constructs from critical ischemia and to form a functional vascular network in vivo. Based on this context and in order to overcome the limitations concerning the isolation and expansion of human SMCs, we developed a protocol to differentiate human pluripotent stem cells (hPSCs) to SMCs. In addition, we generated ECs from the differentiation of hPSCS (hPSC-ECs). hPSCs-SMCs and ECs (hPSC-ECs or primary ECs) were then used to generate 3D vascular organoids which rapidly give rise to a complex three-dimensional vascular network.
METHODOLOGY: We developed an in vitro approach to induce the differentiation of hPSCs (human embryonic stem cells and human induced pluripotent stem cells) to defined SMC populations (contractile and synthetic hPSC-SMCs) using feeder-free and low serum conditions. hPSC-SMCs phenotypes and hPSC-ECs were extensively characterized concerning their phenotype and function. Subsequently, using a methylcellulose-based hydrogel system, we generated spheroids consisting of 1,000 cells (ECs and hPSC-SMC) (vascular organoids). Vascular organoids were extensively characterized regarding their phenotype, cell-cell interactions and their ability to form a three-dimensional capillary network in vitro. 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.
RESULTS: hPSC-SMCs were phenotypically and functionally stable for at least 8 passages and had the ability to stabilize vessel formation and inhibit network regression, when co-cultured with hPSC-ECs in vitro. Furthermore, hPSC-EC/hPSC-SMCs vascular organoids served as focal starting points for the sprouting of capillary-like structures in vitro, using defined matrices of extracellular matrix components.
CONCLUSIONS: We developed a robust method for the generation of defined hSMCs phenotypes from hPSCs. In addition, we differentiated hPSCs to ECs. Fabrication of hECs/hPSC-SMCs vascular organoids embedded in chemically defined matrices is a significant step forward in tissue engineering and regenerative medicine.
*This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Program «Human Resources Development, Education and Lifelong Learning 2014- 2020» in the context of the project “Generation of distinct phenotypes of mural cells from differentiation of human pluripotent stem cells: application in the generation of vascularized tissue engineered constructs” (MIS 5047550)."