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

Skin regeneration with a synthetic biomaterial that replaces the extracellular matrix

Our extracellular matrix replacement (EMR) is a photopolymerized, polysaccharide-PEG hydrogel scaffold that promotes functional skin regeneration and stimulates wound closure with reduced scarring. The advanced wound care market is dominated by collagen-based products isolated from animal and human tissues, and these products are often costly, show only modest efficacy, and cannot be extensively modified to provide customized devices. Preclinical data indicates that the EMR provides the optimal mechanical and chemical properties to stimulate an effective and efficient inflammatory response, followed by regeneration of a robust vascular network and restoration of a complete reticulated epithelium, hair follicles, and sebaceous glands. The EMR is a biodegradable synthetic biomaterial, and its properties can be rationally modified by adjusting the reaction conditions under which it is produced. Therefore, future generations of EMR products can be tailored to specific wound types and/or loaded with growth factors and small molecules. We will review EMR discovery and development, covering its synthesis and chemical and biochemical characterization. We will present data from our preclinical animal studies in murine and porcine third degree burn and excisional wound models. Finally, we will discuss efforts to develop scalable EMR manufacturing processes and highlight some future directions to develop next generation EMR-based products

Hypoxia Affects the Structure of Breast Cancer Cell Derived Matrix to Support Angiogenic Responses of Endothelial Cells

Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDAMB 231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia exposed cocultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report that in vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for understanding how the hypoxic matrix may regulate angiogenesis in tumors

Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells

Control of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the selfrenewal and differentiation of hESCs

Micro-Bioreactor Array for Controlling Cellular Microenvironments

High throughput experiments can be used to spatially and temporally investigate the many factors that regulate cell differentiation. We have developed a micro-bioreactor array (MBA) that is fabricated using soft lithography and contains twelve independent micro-bioreactors perfused with culture medium. The MBA enables cultivation of cells that are either attached to substrates or encapsulated in hydrogels, at variable levels of hydrodynamic shear, and with automated image analysis of the expression of cell differentiation markers. The flow and mass transport in the MBA were characterized by computational fluid dynamic (CFD) modeling. The representative MBA configurations were validated using the C2C12 cell line, primary rat cardiac myocytes and human embryonic stem cells (hESCs) (lines H09 and H13). To illustrate the utility of the MBA for controlled studies of hESCs, we established correlations between the expression of smooth muscle actin and cell density for three different flow configurations

Vascular Progenitor Cells Isolated from Human Embryonic Stem Cells

Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development

Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade

Biophysical regulation of vascular differentiation and assembly

This book updates and expands on various aspects of the vasculature’s microenvironment and how these regulate differentiation and assembly. Discussed in this new edition are efforts to capitalize on combining engineering techniques, to study and manipulate various biophysical cues, including: endothelial cell-pericyte interactions (Davis), mechanical forces to regulate vascularization in three-dimensional constructs (Levenberg), how matrix properties and oxygen tension regulate vascular fate and assembly (Gerecht), biophysical cues in relation to vascular aging (Ferreira), 3D printing of complex vascularized tissue (Hibino), the harnessing of biophysical cues for therapeutic vasculature interfacing with the damaged brain (Segura) and finally, the infarcted heart (Grayson). This second edition of Biophysical Regulation of Vascular Differentiation and Assembly provides an interdisciplinary view of vasculature regulation through various biophysical cues and presents recent advances in measuring and controlling such parameters. This book will be of interest to biologists, biophysicists and engineers who work with vascular differentiation and assembly

Biophysical Regulation of Vascular Differentiation and Assembly

The ability to grow stem cells in the laboratory and to guide their maturation to functional cells allows us to study the underlying mechanisms that govern vasculature differentiation and assembly in health and disease. Accumulating evidence suggests that early stages of vascular growth are exquisitely tuned by biophysical cues from the microenvironment, yet the scientific understanding of such cellular environments is still in its infancy. Comprehending these processes sufficiently to manipulate them would pave the way to controlling blood vessel growth in therapeutic applications. This book assembles the works and views of experts from various disciplines to provide a unique perspective on how different aspects of its microenvironment regulate the differentiation and assembly of the vasculature. In particular, it describes recent efforts to exploit modern engineering techniques to study and manipulate various biophysical cues. Biophysical Regulation of Vascular Differentiation and Assembly provides an interdisciplinary view of vasculature regulation by various biophysical cues and presents recent advances in measuring and controlling such parameters. This book will be of interest to biologists, biophysicists and engineers who work with vascular differentiation and assembly