Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1) , vascular endothelial growth factor-D (VEGF-D) , and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC ^CM resulted in increased tubulogenic efficiency compared with that observed in DPC ^CM . Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.

To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.

Adult mesenchymal stem cells secrete a variety of angiogenic cytokines and growth factors, so we proposed that these paracrine mechanisms may be used to promote vascularization and growth for tissue engineering in vivo. We tested whether or not human adipose-derived stem cells (ASCs) promote tissue formation in rats. ASCs were evaluated in vitro for mRNA expression of angiogenic factors, including the vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 (IL-8), and stromal cell-derived factor-1 (SDF-1) and proliferative activity on human microvascular endothelial cells. For in vivo analysis, CM-DiI-labeled ASCs were implanted with a rat cardiac extracellular matrix (ECM) extract-derived hydrogel into a chamber with a femoral arteriovenous loop in the groin of male nude rats for 7 days. Vascularization in newly generated tissue was estimated by histomorphometry after endothelial nitric oxide synthase (eNOS) immunostaining. ASCs expressed growth factor mRNA and produced an angiogenic activity in vitro. After implantation, ASCs survived, but remained suspended in the ECM and relatively few were incorporated into the newly formed tissue. The volume of newly generated tissue was significantly higher in chambers containing ASCs and it was enriched with vasculature when compared with the ECM alone. We conclude that human ASCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue-engineering applications for regenerative therapy.

Human adipose-derived stem cells (ASCs) secrete cytokines and growth factors that can be harnessed in a paracrine fashion for promotion of angiogenesis, cell survival, and activation of endogenous stem cells. We recently showed that hypoxia is a powerful stimulus for an angiogenic activity from ASCs in vitro and here we investigate the biological significance of this paracrine activity in an in vivo angiogenesis model. A single in vitro exposure of ASCs to severe hypoxia (<0.1% O ₂ ) significantly increased both the transcriptional and translational level of the vascular endothelial growth factor-A (VEGF-A) and angiogenin (ANG). The angiogenicity of the ASC-conditioned medium (ASC ^CM ) was assessed by implanting ASC ^CM -treated polyvinyl alcohol sponges subcutaneously for 2 weeks in mice. The morphometric analysis of anti-CD31-immunolabeled sponge sections demonstrated an increased angiogenesis with hypoxic ASC ^CM treatment compared to normoxic control ASC ^CM treatment (percentage vascular volume; 6.0%±0.5% in the hypoxic ASC ^CM vs. 4.1%±0.7% in the normoxic ASC ^CM , P <0.05). Reduction of VEGF-A and ANG levels in the ASC ^CM with respective neutralizing antibodies before sponge implantation showed a significantly diminished angiogenic response (3.5%±0.5% in anti-VEGF-A treated, 3.2%±0.7% in anti-ANG treated, and 3.5%±0.6% in anti-VEGF-A/ANG treated). Further, both the normoxic and hypoxic ASC ^CM were able to sustain in vivo lymphangiogenesis in sponges. Collectively, the model demonstrated that the increased paracrine production of the VEGF-A and ANG in hypoxic-conditioned ASCs in vitro translated to an in vivo effect with a favorable biological significance. These results further illustrate the potential for utilization of an in vitro optimized ASC ^CM for in vivo angiogenesis-related applications as an effective cell-free technology.