Donny Hanjaya-Putra

Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix

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 (ECFCs) to form efficient vascular networks. Entrapped ECFCs underwent tubulogenesis dependent on the cellular interactions with the HA hydrogel during each stage of vascular morphogenesis. Vacuole and lumen formed through integrins α(5)β(1) and α(V)β(3), while branching and sprouting were enabled by HA hydrogel degradation. Vascular networks formed within HA hydrogels containing ECFCs anastomosed with the hosts circulation and supported blood flow in the hydrogel after transplantation. Collectively, we show that the signaling pathways of vascular morphogenesis of ECFCs can be precisely regulated in a synthetic matrix, resulting in a functional microvasculature useful for the study of 3-dimensional vascular biology and toward a range of vascular disorders and approaches in tissue regeneration.

Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix

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.

Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

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.

Spatial control of cell-mediated degradation to regulate vasculogenesis and angiogenesis in hyaluronan hydrogels.

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.

Smooth-Muscle-Like Cells Derived from Human Embryonic Stem Cells Support and Augment Cord-Like Structures In Vitro

Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-β1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers—including α-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain—to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

Vascular engineering using human embryonic stem cells

Engineering vascularized tissue constructs remains a major problem in regenerative medicine. The formation of such a microvasculature—like the vasculogenesis in early embryogenesis that it closely resembles—is guided by biochemical and biophysical cues, such as growth factors, extracellular matrix proteins, hypoxia, and hydrodynamic shear. As they undergo spontaneous and directed vascular differentiation, human embryonic stem cells can be used as a model system to explore central issues in engineering vascularized tissue constructs and, potentially, to elucidate vasculogenic and angiogenic mechanisms involved in such vascular diseases as limb and cardiac ischemia. Because the conventional spontaneous differentiation approach can only isolate small quantities of vascular cells, recent efforts have sought to develop controlled approaches, including the development of three‐dimensional scaffolds to reengineer the microenvironments of early embryogenesis. This review focuses on emerging approaches to deriving and directing vasculatures from human embryonic stem cells and efforts to engineer 3D vasculatures from such derivatives.

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells.

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape.

Integration and Regression of Implanted Engineered Human Vascular Networks During Deep Wound Healing

The ability of vascularized constructs to integrate with tissues may depend on the kinetics and stability of vascular structure development. This study assessed the functionality and durability of engineered human vasculatures from endothelial progenitors when implanted in a mouse deep burn‐wound model. Human vascular networks, derived from endothelial colony‐forming cells in hyaluronic acid hydrogels, were transplanted into third‐degree burns. On day 3 following transplantation, macrophages rapidly degraded the hydrogel during a period of inflammation; through the transitions from inflammation to proliferation (days 5–7), the hosts vasculatures infiltrated the construct, connecting with the human vessels within the wound area. The growth of mouse vessels near the wound area supported further integration with the implanted human vasculatures. During this period, the majority of the vessels (∼60%) in the treated wound area were human. Although no increase in the density of human vessels was detected during the proliferative phase, they temporarily increased in size. This growth peaked at day 7, the middle of the proliferation stage, and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the vasculatures including the transplanted human vessels generally regressed, and few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 μm2 (including the human ones), remained in the healed wound. Overall, this study offers useful insights for the development of vascularization strategies for wound healing and ischemic conditions, for tissue‐engineered constructs, and for tissue regeneration.

Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo

Blockade of P-selectin/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis, and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogs. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-selectin with nanomolar affinity (Kd ~ 22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-selectin/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

Mending the Failing Heart with a Vascularized Cardiac Patch

Functional, stem-cell-containing cardiac grafts will require vascularized myocardial constructs to support their survival and integration into the host vasculature. Recently in Tissue Engineering, Part A, Lesman et al. (2009) reported the successful integration of vascular cells and hESC-derived cardiomyoctyes into stable grafts in rat recipients.