Vascular morphogenesis of adipose-derived stem cells is mediated by heterotypic cell-cell interactions.
ABSTRACT Adipose-derived stromal/stem cells (ASCs) are a promising cell source for vascular-based approaches to clinical therapeutics, as they have been shown to give rise to both endothelial and perivascular cells. While it is well known that ASCs can present a heterogeneous phenotypic profile, spontaneous interactions among these subpopulations that result in the formation of complex tissue structures have not been rigorously demonstrated. Our study reports the novel finding that ASCs grown in monolayers in the presence of angiogenic cues are capable of self-assembling into complex, three-dimensional vascular structures. This phenomenon is only apparent when the ASCs are seeded at a high density (20,000 cells/cm(2)) and occur through orchestrated interactions among three distinct subpopulations: CD31-positive cells (CD31+), α-smooth muscle actin-positive cells (αSMA+), and cells that are unstained for both these markers (CD31-/αSMA-). Investigations into the kinetics of the process revealed that endothelial vessel-like structures initially arose from individual CD31+ cells through proliferation and their interactions with CD31-/αSMA- cells. During this period, αSMA+ cells proliferated and appeared to migrate toward the vessel structures, eventually engaging in cell-cell contact with them after 1 week. By 2 weeks, the lumen-containing CD31+ vessels grew greater than a millimeter in length, were lined with vascular basement membrane proteins, and were encased within a dense, three-dimensional cluster of αSMA+ and CD31-/αSMA- cells. The recruitment of αSMA+ cells was largely due to platelet-derived growth factor (PDGF) signaling, as the inhibition of PDGF receptors substantially reduced αSMA+ cell growth and vessel coverage. Additionally, we found that while hypoxia increased endothelial gene expression and vessel width, it also inhibited the growth of the αSMA+ population. Together, these findings underscore the potential use of ASCs in forming mature vessels in vitro as well as the need for a further understanding of the heterotypic interactions among ASC subpopulations.
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ABSTRACT: Vasculature is essential to the functional integration of a tissue-engineered bone graft to enable sufficient nutrient delivery and viability after implantation. Native bone and vasculature develop through intimately coupled, tightly regulated spatiotemporal cell-cell signaling. The complexity of these developmental processes has been a challenge for tissue engineers to recapitulate, resulting in poor co-development of both bone and vasculature within a unified graft. To address this, we cultured adipose-derived stromal/stem cells (ASCs), a clinically relevant, single cell source that has been previously investigated for its ability to give rise to vascularized bone grafts, and studied the effects of initial spatial organization of cells, the temporal addition of growth factors, and the presence of exogenous platelet-derived growth factor-BB (PDGF-BB) on the co-development of bone and vascular tissue structures. Human ASCs were aggregated into multicellular spheroids via the hanging drop method prior to encapsulation and subsequent outgrowth in fibrin gels. Cellular aggregation substantially increased vascular network density, interconnectivity, and pericyte coverage compared to monodispersed cultures. To form robust vessel networks, it was essential to culture ASCs in purely vasculogenic medium for at least 8 days prior to the addition of osteogenic cues. Physiologically relevant concentrations of exogenous PDGF-BB (20 ng/ml) substantially enhanced both vascular network stability and osteogenic differentiation. Comparisons with bone morphogenetic protein-2 (BMP-2), another pro-osteogenic and pro-angiogenic growth factor, indicated that this potential to couple the formation of both lineages might be unique to PDGF-BB. Furthermore, the resulting tissue structure demonstrated the close association of mineral deposits with pre-existing vascular structures that have been described for developing tissues. This combination of a single cell source with a potent induction factor used at physiological concentrations can provide a clinically relevant approach to engineering highly vascularized bone grafts.Tissue Engineering Part A 04/2013; · 4.64 Impact Factor
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ABSTRACT: Adipose-derived stem/stromal cell (ASC)-based tissue engineered muscle grafts could provide an effective alternative therapy to autografts - which are limited by their availability - for the regeneration of damaged muscle. However, the current myogenic potential of ASCs is limited by their low differentiation efficiency into myoblasts. The aim of this study was to enhance the myogenic response of human ASCs to biochemical cues by providing biophysical stimuli (11% cyclic uniaxial strain, 0.5 Hz, 1h/day) to mimic the cues present in the native muscle microenvironment. ASCs elongated and fused upon induction with myogenic induction medium alone. Yet, their myogenic characteristics were significantly enhanced with the addition of biophysical stimulation; the nuclei per cell increased approximately 4.5 fold by day 21 in dynamic compared to static conditions (23.3 ± 7.3 vs. 5.2 ± 1.6, respectively), they aligned at almost 45° to the direction of strain, and exhibited significantly higher expression of myogenic proteins (desmin, myoD and myosin heavy chain). These results demonstrate that mimicking the biophysical cues inherent to the native muscle microenvironment in monolayer ASC cultures significantly improves their differentiation along the myogenic lineage.Biochemical and Biophysical Research Communications 07/2013; · 2.41 Impact Factor
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ABSTRACT: We used a combination of strategies to stimulate the vascularization of tissue engineered constructs in vivo including a modular approach to build larger tissues from individual building blocks ('modules') mixed together. Each building block included vascular cells by design: modules were submillimeter-sized collagen gels with an outer layer of endothelial cells (EC), and with embedded adipose-derived mesenchymal stem cells (adMSC) to support EC survival and blood vessel maturation in vivo. We transduced the EC coating the modules with a lentiviral construct to overexpress the angiogenic extracellular matrix (ECM) protein Developmental endothelial locus-1 (Del-1). Upon injection of modules in a subcutaneous SCID/Bg mouse model, there was an increase in the number of blood vessels for implants with EC transduced to overexpress Del-1 compared to control implants (with eGFP transduced EC) over the 21 day duration of the study. The greatest difference between Del-1 and eGFP implants and the highest number of blood vessels was observed seven days after transplantation. The day 7 Del-1 implants also had increased SMA+ staining compared to control, suggesting increased blood vessel maturation through recruitment of SMA+ smooth muscle cells or pericytes to stabilize the newly formed blood vessels. Perfusion studies (microCT, ultrasound imaging, and systemic injection of fluorescent UEA-1 or dextran) showed that some of the newly formed blood vessels (both donor-derived and host-derived, in both Del-1 and eGFP implants) were perfused and connected to the host vasculature as early as seven days after transplantation, and at later time points as well. Nevertheless, perfusion of the implants was limited in some cases, suggesting further improvements are necessary to normalize the vasculature at the implant site.Tissue Engineering Part A 10/2013; · 4.64 Impact Factor