The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.codifferentiation | hydrogels

Understanding the role of the extracellular matrix (ECM) in vascular morphogenesis has been possible using natural ECMs as in vitro models to study the underlying molecular mechanisms. However, little is known about vascular morphogenesis in synthetic matrices where properties can be tuned toward both the basic understanding of tubulogenesis in modular environments and as a clinically relevant alternative to natural materials for regenerative medicine. We investigated synthetic, tunable hyaluronic acid (HA) hydrogels and determined both the adhesion and degradation parameters that enable human endothelial colony-forming cells ( IntroductionGenerating a functional vascular network can potentially improve treatment for vascular disease and successful organ transplantation. 1 Since their discovery, marrow-derived circulating endothelial progenitor cells (EPCs) have been demonstrated to participate in postnatal vasculogenesis. 2,3 Putative EPCs have been proposed as a potential therapeutic tool for treating vascular disease, either through infusion to the site of vascularization [4][5][6] or via ex vivo expansion to engineer vascularized tissue constructs. [7][8][9] Research has shown that endothelial colony-forming cells (ECFCs), a subtype of EPCs recently identified from circulating adult and human umbilical cord blood, express characteristics of putative EPCs. 10,11 These ECFCs are characterized by robust proliferative potential in forming secondary and tertiary colonies, as well as de novo blood vessel formation in vivo.The complex processes of vascular regeneration and repair require EPCs to break down the extracellular matrix (ECM), migrate, differentiate, and undergo tubulogenesis. In the last decade, our understanding of the role of the ECM in vascular morphogenesis has greatly expanded because of well-defined in vitro angiogenesis models. Such natural ECMs as matrigel, collagen, and fibrin gels have allowed us to study the molecular mechanisms that regulate endothelial cell (EC) tubulogenesis, 12,13 as well as to transplant vascular progenitor cells, such as human embryonic stem (hES) cell-derived ECs, 14 ECFCs, 15 EPCs, and mesenchymal stem cells (MSCs), 8,9,16 to generate vascular networks and in vivo. However, the inherent chemical and physical properties of these natural materials have limited their manipulability for engineering vascularized tissue constructs. Moreover, problems associated with complex purification processes, pathogen transfer, and immunogenicity have hampered their clinical usage. 17 Some have suggested synthetic biomaterials, xeno-free and more clinically relevant for regenerative medicine, as an alternative. 18 Unlike natural ECMs, we can engineer these synthetic biomaterials to provide instructive microenvironments capable of recapitulating complex stages of vascular morphogenesis. 17 Although several studies have attempted to generate vascular network assembly within such synthetic biomaterials in vitro, 19,20 no report to date demonstrates highly controlled vascular morphogenes...

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co-regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1-MMP (MT1-MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.

Matrix remodeling is crucial for neovascularization, however its utilization to control this process in synthetic biomaterials has been limited. Here, we utilized hyaluronic acid (HA) hydrogels to spatially control cellular remodeling during vascular network formation. Specifically, we exploited a secondary radical polymerization to alter the ability of cells to degrade the hydrogel and utilized it to create spatial patterning using light initiation. We first demonstrated the ability of the hydrogel to either support or inhibit in vitro vasculogenesis of endothelial colony-forming cells (ECFCs) or angiogenesis from ex ovo chorioallantoic membranes. We showed that vascular tube branching and sprouting, which required matrix metalloproteinases (MMPs)-dependent remodeling, could be achieved in hydrogels formed by primary addition-crosslinking only. Although ECFCs expressed higher levels of MMPs in the hydrogels with the secondary radical-crosslinking, the generated kinetic chains disabled cell-mediated remodeling and therefore vascular formation was arrested at the vacuole and lumen stage. We then patterned hydrogels to have regions that either permitted or inhibited cell-mediated degradation during in vitro vasculogenesis or angiogenesis. Our ability to control degradation cues that regulate vascular tube formation is important for the study of vascular biology and the application of synthetic biomaterials in tissue regeneration.