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.
SourceAvailable from:[Show abstract] [Hide abstract]
ABSTRACT: The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue in vitro with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both in vitro and in vivo. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.PLoS ONE 09/2014; 9(9):e107199. DOI:10.1371/journal.pone.0107199 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Rationale: Adipose stromal cells (ASC) are therapeutically potent progenitor cells that possess properties of pericytes. In vivo, ASC in combination with endothelial cells (EC) establish functional multilayer vessels, in which ASC form the outer vessel layer and differentiate into mural cells. Objective: To identify factors responsible for ASC differentiation towards the smooth muscle cell (SMC) phenotype via interaction with EC. Methods and Results: An in vitro model of EC co-cultivation with ASC was employed, in which EC organized into vascular cords, accompanied by ASC migration towards EC and up-regulation of αSMA, SM22α, and calponin expression. Conditioned media (CM) from EC-ASC, but not from EC cultures, induced SMC protein expression in ASC monocultures. EC-ASC co-cultivation induced marked accumulation of activin A, but not TGFβ1 in CM. This was attributed to induction of activin A expression in ASC upon contact with EC. While TGFβ and activin A were individually sufficient to initiate expression of SMC antigens in ASC, only activin A IgG blocked the effect of EC-ASC CM. While TGFβ was able to induce activin A expression in ASC, in co-cultures this induction was TGFβ-independent. In EC-ASC co-cultures activin A IgG or ALK4/5/7 receptor inhibitors blocked expression of αSMA in ASC in the absence of direct EC-cord contact, but this inhibition was circumvented in ASC by direct EC contact. Conclusions: EC initiate a SMC differentiation program in adjacent ASC, and propagate this differentiation in distant ASC, by induction of activin A expression.Circulation Research 08/2014; 115(9). DOI:10.1161/CIRCRESAHA.115.304026 · 11.09 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Trabecular bone has an interconnected porous structure, which influences cellular responses, biochemical transport and mechanical strength. Appropriately mimicking this structural organization in biomaterial scaffolds can facilitate more robust bone tissue regeneration and integration by providing a native microenvironment to the cells. This study examined the effect of pore size on human adipose-derived stem/stromal cell (ASC) osteogenesis within poly(ε-caprolactone) (PCL) scaffolds. Scaffold pore size was controlled by porogen leaching of custom-made paraffin particles with three different size ranges: P200 (< 500 µm), P500 (500-1000 µm), and P1000 (1000-1500 µm). Scaffolds produced by leaching these particles exhibited highly interconnected pores and rough surface structures that were favorable for cell attachment and ingrowth. The osteogenic response of ASCs was evaluated following 3 weeks of in vitro culture using biochemical (ALP, Ca(2+)/DNA content), mechanical (compression test) and histological (H&E and von Kossa staining) analyses. It was observed that while the total number of cells was similar for all scaffolds, the cell distributions and osteogenic properties were affected by the scaffold pore size. ASCs were able to bridge smaller pores and grow uniformly within these scaffolds (P200) while they grew as a layer along the periphery of the largest pores (P1000). The cell-biomaterial interactions specific to the latter case led to enhanced osteogenic responses. The ALP activity and Ca(2+) deposition were doubled in P1000 scaffolds as compared to P200 scaffolds. A significant difference was observed between the compressive strength of unseeded and seeded P1000 scaffolds. Therefore, we demonstrated that the use of scaffolds with pores that are in the range of 1 mm enhances in vitro ASC osteogenesis, which may improve their performance in engineered bone substitutes.Biomedical Materials 06/2014; 9(4):045003. DOI:10.1088/1748-6041/9/4/045003 · 2.92 Impact Factor