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...

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid beta-glucocerebrosidase (GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the source of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and if their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human iPS cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of TNF alpha, IL-6, and IL-1beta than control cells, and this phenotype was exacerbated by treatment with LPS. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of RBC remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant glucocerebrosidase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients, and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery.

The generation of functional vascular networks has the potential to improve treatment for vascular diseases and to facilitate successful organ transplantation. Endothelial colony-forming cells (ECFCs) have robust proliferative potential and can form vascular networks in vivo. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix instigate vascular morphogenesis. Although this process has been elucidated using natural matrix, little is known about vascular morphogenesis by ECFCs in synthetic matrix, a xeno-free scaffold that can provide a more controllable and clinically relevant alternative for regenerative medicine. We sought to study hyaluronic acid (HA) hydrogels as three-dimensional scaffolds for capillary-like structure formation from ECFCs, and to determine the crucial parameters needed to design such synthetic scaffolds. We found that ECFCs express HA-specific receptors and that vascular endothelial growth factor stimulates hyaluronidase expression in ECFCs. Using a well-defined and controllable three-dimensional HA culture system, we were able to decouple the effect of matrix viscoelasticity from changes in adhesion peptide density. We determined that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of vascular endothelial growth factor within the hydrogel depends on the makeup of the synthetic environment. Collectively, these results set forth initial design criteria that need to be considered in developing vascularized tissue constructs.

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