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Supplementary Figure 6 from White Adipose Tissue Cells Are Recruited by Experimental Tumors and Promote Cancer Progression in Mouse Models

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Aging is associated with an increased susceptibility to adverse inflammatory conditions such as sepsis and cytokine storm. We hypothesized that senescent cells (SnCs) play a central role in this age-associated pathology in part due to their expression of the senescence-associated secretory phenotype (SASP), which may prime SnCs to inflammatory stimulation. To test this hypothesis, we examined the expression of various inflammatory cytokines and chemokines at the levels of gene transcription and protein production in various SnCs in vitro in response to lipopolysaccharide (LPS), interleukin-1β (IL1β), and tumor necrosis factor α (TNFα) stimulation. We found that SnCs not only expressed higher basal levels of various inflammatory cytokines and chemokines as a manifestation of the SASP, but more importantly exhibited hyper-activation of the induction of a variety of inflammatory mediators in response to LPS, IL1β and TNFα stimulation as compared with non-SnCs. This senescence-associated hyper-activation is likely mediated in part via the p38MAPK (p38) and NFκB pathways because LPS stimulation elicited significantly higher levels of p38 phosphorylation and NFκB p65 nuclear translation in SnCs when compared to their non-senescent counterparts and inhibition of these pathways with losmapimod (a p38 specific inhibitor) and BMS-345541 (a selective NFκB inhibitor) attenuated LPS-induced expression of IL6, TNFα, CCL5, and IL1β mRNA in SnCs. These findings suggest that SnCs may play an important role in the age-related increases in the susceptibility to developing an exacerbated inflammatory response and highlight the potential to use senotherapeutics to ameliorate the severity of various devastating inflammatory conditions in the elderly.

Heart transplantation is a life-saving therapy for end-stage organ failure. Organ deterioration during transportation limits storage to 4 hours, limiting hearts available. Approaches ameliorating organ damage could increase the number of hearts acceptable for transplantation. Prior studies show that adipose-derived stem/stromal cell secretome (ASC-S) rescues tissues from postischemic damage in vivo. This study tested whether ASC-S preserved the function of mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iCM) exposed to organ transportation and transplantation conditions. Hearts were subjected to cold University of Wisconsin (UW) cardioplegic solution ± ASC-S for 6 hours followed by analysis using the Langendorff technique. In parallel, the effects of ASC-S on the recovery of iCM from UW solution were examined when provided either during or after cold cardioplegia. Exposure of hearts and iCM to UW deteriorated contractile activity and caused cell apoptosis, worsening in iCM as a function of exposure time; these were ameliorated by augmenting with ASC-S. Silencing of superoxide dismutase 3 and catalase expression prior to secretome generation compromised the ASC-S cardiomyocyte-protective effects. In this study, a novel in vitro iCM model was developed to complement a rodent heart model in assessing efficacy of approaches to improve cardiac preservation. ASC-S displays strong cardioprotective activity on iCM either with or following cold cardioplegia. This effect is associated with ASC-S-mediated cellular clearance of reactive oxygen species. The effect of ASC-S on the temporal recovery of iCM function supports the possibility of lengthening heart storage by augmenting cardioplegic transport solution with ASC-S, expanding the pool of hearts for transplantation.

Aim: to evaluate the impact of tissue-engineered structures (TES) transplantation based on mesenchymal stromal cell (MSC) sheets in myocardial infarction on the activation of the epicardial cell pool and vascularization of the damaged zone.Materials and methods. Mesenchymal stromal cells were obtained from samples of subcutaneous fat of Wistar rats and C57Bl/6 mice. Tissue engineering structures were obtained by culturing cell sheets on thermosensitive plates (Nunc Dishes with UpCell Surface). Transplantation of TESs was performed after myocardial infarction modeling in rats by ligation of the anterior descending coronary artery. Transplant cells and damaged zones were assessed using immunofluorescent staining of myocardial cryosections. The impact of MSC secretion products on the migration activity of epicardial cells in vitro was evaluated using the explant culture method.Results. MSCs in TESs after transplantation remain viable and induce activation of the epicardial cell pool and local increase of the damaged zone vascularization. The in vitro experiments showed that the conditioned environment of MSCs stimulates the migratory activity of epicardial cells and initiates the formation of activated Wt1/POD1 precursor cells.Conclusion. TES transplantation on the basis of MSC sheets seems to be a promising approach for effective delivery of viable cells into myocardium to activate the epicardial cellular niche and reparative angiogenesis.

Acute ischaemia causes a significant loss of blood vessels leading to deterioration of organ function. Multiple ischaemic conditions are associated with up‐regulation of activin A, but its effect on endothelial cells (EC) in the context of hypoxia is understudied. This study evaluated the role of activin A in vasculogenesis in hypoxia. An in vitro vasculogenesis model, in which EC were cocultured with adipose stromal cells (ASC), was used. Incubation of cocultures at 0.5% oxygen led to decrease in EC survival and vessel density. Hypoxia up‐regulated inhibin BA (monomer of activin A) mRNA by 4.5‐fold and activin A accumulation in EC‐conditioned media by 10‐fold, but down‐regulated activin A inhibitor follistatin by twofold. Inhibin BA expression was also increased in human EC injected into ischaemic mouse muscles. Activin A secretion was positively modulated by hypoxia mimetics dimethyloxalylglycine and desferrioxamine. Silencing HIF1α or HIF2α expression decreased activin A secretion in EC exposed to hypoxia. Introduction of activin A to cocultures decreased EC number and vascular density by 40%; conversely, blockade of activin A expression in EC or its activity improved vasculogenesis in hypoxia. Activin A affected EC survival directly and by modulating ASC paracrine activity leading to diminished ability of the ASC secretome to support EC survival and vasculogenesis. In conclusion, hypoxia up‐regulates EC secretion of activin A, which, by affecting both EC and adjacent mesenchymal cells, creates a micro‐environment unfavourable for vasculogenesis. This finding suggests that blockade of activin A signalling in ischaemic tissue may improve preservation of the affected tissue.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31−/CD45−/CD34+/CD146− cells (adventitial stromal/stem cells [ASCs]) and CD31−/CD45−/CD34−/CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbrASC (most primitive); (b) ALDHdimASC; (c) ALDHbrPC; (d) ALDHdimPC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks.

No specific therapy exists for acute pancreatitis (AP), and current treatment remains entirely supportive. Adipose stem cells (ASCs) have significant immunomodulatory and regenerative activities. We hypothesized that systemic administration of ASCs would mitigate inflammation in AP. AP was induced in mice by 6 hourly intraperitoneal injections of cerulein. Twenty-four hours after AP induction, mice were randomized into four systemic treatment groups: sham group (no acute pancreatitis), vehicle, human ASCs, and human ASC–conditioned media. Mice were sacrificed at 48 h, and blood and organs were collected and analyzed. Pancreatic injury was quantified histologically using a published score (edema, inflammation, and necrosis). Pancreatic inflammation was also studied by immunohistochemistry and PCR. When using IV infusion of Hoechst-labeled ASCs, ASCs were found to localize to inflamed tissues: lungs and pancreas. Mice treated with ASCs had less severe AP, as shown by a significantly decreased histopathology score (edema, inflammation, and necrosis) (p = 0.001). ASCs infusion polarized pancreatic macrophages toward an anti-inflammatory M2 phenotype. ASC-conditioned media reduced pancreatic inflammation similarly to ASCs only, highlighting the importance of ASCs secreted factors in modulating inflammation. Intravenous delivery of human ASCs markedly reduces pancreatic inflammation in a murine model of AP ASCs which represent an effective therapy for AP.

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell-contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC-lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ1 ) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre-differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre-differentiated ASCs were used. Pre-differentiated ASCs had an inferior mitogenic response to EC-produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co-culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFβ1 induced secretion of activin A. Augmenting co-culture incubation media with anti-activin A IgG restored the ability of pre-differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFβ1 or activin A-induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co-injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS–exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity.

Objectives The potential for beneficial effects of adipose-derived stem cells (ASCs) on myocardial perfusion and left ventricular dysfunction in myocardial ischemia (MI) has not been tested following intravenous delivery. Methods Surviving pigs following induction of MI were randomly assigned to 1 of 3 different groups: the placebo group (n = 7), the single bolus group (SB) (n = 7, 15 × 107 ASCs), or the divided dose group (DD) (n = 7, 5 × 107 ASCs/day for three consecutive days). Myocardial perfusion defect area and coronary flow reserve (CFR) were compared during the 28-day follow-up. Also, serial changes in the absolute number of circulating CD4+T and CD8+T cells were measured. Results The increases in ejection fraction were significantly greater in both the SB and the DD groups compared to the placebo group (5.4 ± 0.9%, 3.7 ± 0.7%, and −0.4 ± 0.6%, respectively), and the decrease in the perfusion defect area was significantly greater in the SB group than the placebo group (−36.3 ± 1.8 and −11.5 ± 2.8). CFR increased to a greater degree in the SB and the DD groups than in the placebo group (0.9 ± 0.2, 0.8 ± 0.1, and 0.2 ± 0.2, respectively). The circulating number of CD8+T cells was significantly greater in the SB and DD groups than the placebo group at day 7 (3,687 ± 317/µL, 3,454 ± 787/µL, and 1,928 ± 457/µL, respectively). The numbers of small vessels were significantly greater in the SB and the DD groups than the placebo group in the peri-infarct area. Conclusions Both intravenous SB and DD delivery of ASCs are effective modalities for the treatment of MI in swine. Intravenous delivery of ASCs, with its immunomodulatory and angiogenic effects, is an attractive noninvasive approach for myocardial rescue. © 2014 Wiley Periodicals, Inc.

Objective: Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays. Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion: Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression. Stem Cells 2015;33:468–478

The interaction between human endothelial (EC) and adipose mesenchymal stem/stromal cells (ASC) leads to EC reorganization into stable vascular structures in vitro and functional vessels in vivo and ASC maturation from progenitor to smooth muscle/pericyte phenotype. We have previously shown that juxtacrine EC-ASC interactions induce in ASC to express activin A, which, by affecting both EC and ASC bioactivities, plays a central role in vasculogenesis. Here, a co-culture model of human cord blood-derived EC and human ASC was used to study the role of Notch2 and Notch3 in juxtacrine mechanism-mediated regulation of activin A expression in ASC. Blockade of Notch signaling by the γ-secretase inhibitor DAPT, between days 1-3 following heterocellular contact, prevented induction of activin A secretion, and DAPT added to co-cultures after establishment of activin A secretion almost completely abrogated its production, suggesting the need for uninterrupted Notch signaling to enforce continued activin A production. In parallel, DAPT reduced ASC differentiation towards the mural phenotype, and increased EC proliferation, causing inefficient vasculogenesis. Silencing Notch2 expression in ASC prevented activin A production by ASC when in co-culture with EC, whereas silencing Notch3 expression amplified its secretion. Dual silencing experiments revealed that silencing Notch2 was dominant, eliminating the effect of silencing Notch3. In parallel, Notch2 was not involved in mediating differentiation of ASC towards a smooth muscle/pericyte fate, whereas silencing of Notch3 led to an increase in baseline expression of markers of smooth muscle phenotype, which was further enhanced on contact with EC. Overall, Notch3 inhibits both expression of activin A and smooth muscle/pericyte differentiation program. Furthermore, these data suggest that activin A is induced exclusively through trans signaling mechanisms, whereas mural differentiation program is also regulated through cis pathways.

Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short‐term recovery and long‐term functional preservation post‐AKI. Human adipose stromal cells (hASCs) possess pro‐angiogenic and anti‐inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC‐treated rats had significantly greater survival compared to vehicle‐treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post‐AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC‐treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle‐treated rats as well as reduced accumulation of interstitial alpha‐smooth muscle actin‐positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R‐induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.

An increased demand for soft-tissue substitutes has impelled the development of an in vitro adipose tissue. Ideally, such a tissue should contain a vascular network that can deliver blood throughout the construct following its engraftment. This study describes the in vitro fabrication of a pre-vascularized adipose tissue entirely using a self-assembly approach. Adult human adipose stromal cells (ASCs) provided the foundation for this construct. These cells were cultured at high density in the presence of elevated levels of ascorbate prior to adipocytic induction. Vascular support cells consisting of dermal fibroblasts, mixtures of adipose stromal cells and bone marrow mesenchymal stem cells (MSCs) were introduced to sustain an extensive vascular network formed by human umbilical vein endothelial cells (HUVECs). MSCs were introduced to serve as perivascular cells. The resulting construct contained a vascular-adipose tissue continuum that was held together by basement membrane molecules. This construct contains multiple cell types that are typically found in adipose tissue: adipocytes, pre-adipocytes, stem cells, fibroblasts, vascular cells, and perivascular support cells. As such, these constructs can be employed both for in vitro studies to assay cellular interactions between vasculature and other components of adipose tissue. Further, they can also be engrafted into athymic hosts to study vascular and adipocyte stability.

Adipose stromal cells (ASCs) express markers and functional properties of pericytes in vitro and, in combination with endothelial cells (ECs), are able to establish multilayer functional vessels in vivo. However, the factors that coordinate EC-ASC communications to promote migration of these cells toward one another, and their heterotypic assembly into vascular structures are not well defined. To understand the mechanisms of EC-ASC interaction, we developed an in vitro model of coculturing ECs with ASCs in a system containing serum but no additional exogenous cytokines or extracellular matrix (ECM) proteins. We demonstrated that ASCs have a profound potential to stimulate morphogenesis of ECs into branching networks of cord structures. The vascular networks developed in 6 days and were stable for at least 3 weeks. This process was associated with an increase in ECM protein production by ASCs and ECs, alpha-smooth muscle actin expression by ASCs, and increased CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1) surface presentation by ECs. The vascular network formation (VNF) was dependent on matrix metalloproteinase activity and cell communications through vascular endothelial growth factor, hepatocyte growth factor, and platelet-derived growth factor-BB pathways. ASCs exhibited significantly higher potential to stimulate VNF than smooth muscle cells and fibroblasts. Media conditioned by ASCs promoted VNF by ECs cultured on smooth muscle cells and fibroblasts, but could not replace the presence of ASCs in coculture. The presence of ASCs in EC-fibroblast cocultures in a low fraction efficiently stimulated VNF. These findings demonstrate that the vasculogenesis-promoting potential of ASCs depends on interaction with ECs involving contact and likely bi-directional interaction, resulting in modulated secretion of cytokines and ECM proteins.

Rapid induction and maintenance of blood flow through new vascular networks is essential for successfully treating ischemic tissues and maintaining function of engineered neo-organs. We have previously shown that human endothelial progenitor cells (EPCs) form functioning vessels in mice, but these are limited in number and persistence; and also that human adipose stromal cells (ASCs) are multipotent cells with pericytic properties which can stabilize vascular assembly in vitro. In this study, we tested whether ASCs would cooperate with EPCs to coassemble vessels in in vivo implants. Collagen implants containing EPCs, ASCs, or a 4:1 mixture of both were placed subcutaneously into NOD/SCID mice. After a range of time periods, constructs were explanted and evaluated with regard to vascular network assembly and cell fate; and heterotypic cell interactions were explored by targeted molecular perturbations. The density and complexity of vascular networks formed by the synergistic dual-cell system was many-fold higher than found in implants containing either ASCs or EPCs alone. Coimplantation of ASCs and EPCs with either pancreatic islets or adipocytes produced neoorgans populated by these parenchymal cells, as well as by chimeric human vessels conducting flow. This study is the first to demonstrate prompt and consistent assembly of a vascular network by human ASCs and endothelial cells and vascularization by these cells of parenchymal cells in implants. Mixture of these 2 readily available, nontransformed human cell types provides a practical approach to tissue engineering, therapeutic revascularization, and in vivo studies of human vasculogenesis.

The connection between obesity and accelerated cancer progression has been established, but the mediating mechanisms are not well understood. We have shown that stromal cells from white adipose tissue (WAT) cooperate with the endothelium to promote blood vessel formation through the secretion of soluble trophic factors. Here, we hypothesize that WAT directly mediates cancer progression by serving as a source of cells that migrate to tumors and promote neovascularization. To test this hypothesis, we have evaluated the recruitment of WAT-derived cells by tumors and the effect of their engraftment on tumor growth by integrating a transgenic mouse strain engineered for expansion of traceable cells with established allograft and xenograft cancer models. Our studies show that entry of adipose stromal and endothelial cells into systemic circulation leads to their homing to and engraftment into tumor stroma and vasculature, respectively. We show that recruitment of adipose stromal cells by tumors is sufficient to promote tumor growth. Finally, we show that migration of stromal and vascular progenitor cells from WAT grafts to tumors is also associated with acceleration of cancer progression. These results provide a biological insight for the clinical association between obesity and cancer, thus outlining potential avenues for preventive and therapeutic strategies. [Cancer Res 2009;69(12):5259–66]

Human adipose stromal cells (ASCs) reside within the stromal-vascular fraction (SVF) in fat tissue, can be readily isolated, and include stem-like cells that may be useful for therapy. An important consideration for clinical application and functional studies of stem/progenitor cells is their capacity to maintain chromosome stability in culture. In this study, cultured ASC populations and ASC clones were evaluated at intervals for maintenance of chromosome stability. Uncultured SVF (uSVF) cells were included for comparison. G-banded chromosome analysis demonstrated that ASCs are diploid and have a normal karyotype. However since only approximately 20 cells are examined, low levels of chromosome instability would not be detected. To increase detection sensitivity, fluorescence in situ hybridization was employed, to permit chromosome enumeration in larger numbers of interphase cells. Seven cultured ASC populations, two ASC clones and four uSVF samples were examined. Chromosome X and 17 probes identified diploid, tetraploid, and aneuploid interphase cells. Both cultured ASC populations [up to approximately 35 Population Doublings (PDs)] and uSVF cells exhibited a similar level of diploidy (97.8% n = 6,355 and 98.83% n = 1,197, respectively) and numerical abnormalities, suggesting that cultured ASCs are genomically stable and supporting their suitability for transplantation applications. In comparison, cultured primary human chorionic villus cells exhibited marked genomic instability resulting in an 11.6% tetraploidy rate after 8-10 PD. Thus effects of culture on genomic stability may be cell type dependent and should be tested by appropriately scaled interphase fluorescence in situ hybridization analysis in any ex vivo expanded cell population destined for transplantation.

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor produced in human adipose tissue. In this study, we use 3T3-F442A preadipocytes to study the contribution of HGF to angiogenesis in an in vivo fat pad development model. As observed for human adipocytes, HGF is synthesized and secreted by 3T3-F442A preadipocytes and mature adipocytes. HGF knockdown with small-interfering RNA reduced HGF mRNA expression 82.3 ± 4.2% and protein secretion 82.9 ± 1.4% from 3T3-F442A preadipocytes. Silencing of HGF resulted in a 70.5 ± 19.0% reduction in endothelial progenitor cell migration to 3T3-F442A-conditioned medium in vitro. 3T3-F442A preadipocytes injected under the skin of mice form a fat pad containing mature, lipid-filled adipocytes and a functional vasculature. At 72 h postinjection, expression of the endothelial cell genes TIE-1 and platelet endothelial cell adhesion molecule (PECAM)-1 was decreased 94.4 ± 2.2 and 91.5 ± 2.5%, respectively, in 3T3-F442A fat pads with HGF silencing. Knockdown of HGF had no effect on differentiation of 3T3-F442A preadipocytes to mature adipocytes in vitro or in vivo. In developing fat pads under the skin of HGF overexpressing transgenic mice, TIE-1 and PECAM-1 mRNA was increased 16.5- and 21.4-fold, respectively, at 72 h postinjection. The increase in gene expression correlated with immunohistochemical evidence of endothelial cell migration in the developing fat pad. These data suggest that HGF has a central role in regulating angiogenesis in adipose tissue.

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell-contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC-lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ

Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double ( p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent therapeutic cocktail for patients with CLI, many of whom are at risk for amputation of the affected limb.

Background Acute Respiratory Distress Syndrome (ARDS) is a condition that contributes to morbidity and mortality of critically ill patients. We investigated whether factors secreted by adipose stromal cells (ASC) into conditioned media (ASC-CM) will effectively decrease lung injury in the model of lipopolysaccharide (LPS)-induced ARDS. Methods To assess the effect of ASC-CM on ARDS indices, intravenous delivery of ASC and ASC-CM to C57Bl/6 mice was carried out 4 h after LPS oropharyngeal aspiration; Evans Blue Dye (EBD) was injected intravenously 1 h prior to animal sacrifice (48 h post-LPS). Lungs were either fixed for histopathology, or used to extract bronchoalveolar lavage fluid (BALF) or EBD. To assess the effect of ASC-CM on endothelial barrier function and apoptosis, human pulmonary artery endothelial cells were treated with ASC-CM for 48-72 h. Results ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6. White Blood Cells (WBC) from BALF of LPS-challenged mice receiving ASC-CM had decreased reactive oxygen species (ROS) generation compared to WBC from LPS-challenged mice receiving control media injection. Treatment of pulmonary endothelial monolayers with ASC-CM significantly suppressed H2O2-induced leakage of FITC dextran and changes in transendothelial resistance, as well as gap formation in endothelial monolayer. ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3. ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation. Conclusions Factors secreted by ASC efficiently reduce ARDS indices, endothelial barrier hyperpermeability, and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.

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 toward the smooth muscle cell phenotype via interaction with EC. Methods and Results: An in vitro model of EC cocultivation with ASC was used, in which EC organized into vascular cords, accompanied by ASC migration toward EC and upregulation of α-smooth muscle actin, SM22α, and calponin expression. Conditioned media from EC–ASC, but not from EC cultures, induced smooth muscle cell protein expression in ASC monocultures. EC–ASC cocultivation induced marked accumulation of activin A but not transforming growth factor-β1 in conditioned media. This was attributed to induction of activin A expression in ASC on contact with EC. Although transforming growth factor-β and activin A were individually sufficient to initiate expression of smooth muscle cell antigens in ASC, only activin A IgG blocked the effect of EC–ASC conditioned media. Although transforming growth factor-β was able to induce activin A expression in ASC, in cocultures this induction was transforming growth factor-β independent. In EC–ASC cocultures, activin A IgG or ALK4/5/7 receptor inhibitors blocked expression of α-smooth muscle actin 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 smooth muscle cell differentiation program in adjacent ASC and propagate this differentiation in distant ASC by induction of activin A expression.

Adipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in streptozotocin-treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC-derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP-1) to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against β cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects. Stem Cells 2014;32:1831–1842

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved "b" wave amplitude (as measured by electroretinogram) within 1-3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.

Background: The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age. Objectives: We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years. Methods: SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated. Results: The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation. Conclusions: By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved. Level of Evidence: 5 ![Graphic][1] [1]: /embed/inline-graphic-1.gif

Introduction: Autologous bone marrow mesenchymal stromal cells (ABMSCs) have been the focus for the treatment of myocardial and limb ischemia. The utility of ABMSCs is limited because, in addition to requiring an invasive procedure to harvest, the number and the regenerative capacity of ABMSCs decrease with age and comorbid conditions. A redirected focus on alternative sources of MSCs is warranted. Here we compare MSCs from adipose tissue (ASCs) and human placenta (pMSCs) for angiogenic therapies. Methods: ASCs and pMSCs were characterized using culture assays, flow cytometry and immunofluorescent staining. Immunodulatory properties were assessed using mixed lymphocyte reactions (MLR). Inflammatory and angiogenic cytokines were quantified using ELISA. The ability of ASCs and pMSCs to restore perfusion in a murine model of hindlimb ischemia was compared. Results: ASCs and pMSCs expressed cell surface antigens consistent with MSCs. In permissive cultures, ASCs and pMSCs stained positive for Oil Red O (adipogenic), Alcian Blue (chondrogenic) and alkaline phosphatase (osteogenic) in addition to neural and myogenic lineages. Immunofluorescent staining demonstrated that PMSCs but not ASCs express embryonic stem cell antigens Oct-3/4, Nanog, SSEA-3 and SSEA-4. ASCs and pMSCs demonstrated immunosuppressive properties in MLRs (n=3). There was a significant reduction of MNC proliferation when cultured with pMSCs (96.5 ± 3.7 % reduction; p < 0.001) or ASCs (93.27 ± 2.3 % reduction; p < 0.001). When compared to ASCs, pMSCs (n=3) secrete significantly greater quantities of VEGF (4.4 ± 0.13 vs. 2.5 ± 0.031 ng/mL; p < 0.001), HGF (2.4 ± 0.114 vs. 1.6 ± 0.173 ng/mL; p < 0.001), and Ang-1 (4.6 ± .23 vs. 1.6 ± 0.016 ng/mL; p < 0.001) in hypoxia. In NOD/SCID mice with femoral artery ligation (n=6), those treated with pMSCs demonstrated a significant improvement in perfusion compared to those treated with ASCs at 35 days post-treatment (56.18 vs. 40.82; p < 0.001). Conclusion: In conclusion, while both ASCs and pMSCs have potent immunosuppressive properties, pMSCs are superior in the production of angiogenic cytokines and restoration of perfusion in ischemic tissue. PMSCs can be banked and thus made readily available for the treatment of acute ischemic syndromes.

Endothelial colony-forming cells (ECFCs) isolated from umbilical cord blood (CBECFCs) are highly proliferative and form blood vessels in vivo. The purpose of this investigation was to isolate and characterize a population of resident ECFCs from the chorionic villi of term human placenta and provide a comparative analysis of their proliferative and vasculogenic potential with CBECFCs. ECFCs were isolated from umbilical cord blood and chorionic villi from placentas obtained by caesarean deliveries. Placental ECFCs (PECFCs) expressed CD144, CD31, CD105, and KDR and were negative for CD45 and CD34, consistent with other ECFC phenotypes. PECFCs were capable of 28.6 ± 6.0 population doublings before reaching senescence (vs. 47.4 ± 3.2 for CBECFCs, p < 0.05, n = 4). In single cell assays, 46.5 ± 1.2% underwent at least one division (vs. 51.0 ± 1.8% of CBECFCs, p = 0.07, n = 6), and of those dividing PECFCs, 71.8 ± 0.9% gave rise to colonies of >500 cells (highly proliferative potential clones) over 14 days (vs. 69.4 ± 0.7% of CBECFCs, p = 0.07, n = 9). PECFCs formed 5.2 ± 0.8 vessels/mm2 in collagen/fibronectin plugs implanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, whereas CBECFCs formed only 1.7 ± 1.0 vessels/mm2 ( p < 0.05, n = 4). This study demonstrates that circulating CBECFCs and resident PECFCs are identical phenotypically and contain equivalent quantities of high proliferative potential clones. However, PECFCs formed significantly more blood vessels in vivo than CBECFCs, indicating that differences in vasculogenic potential between circulating and resident ECFCs exist.

Rationale: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. Objectives: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. Methods: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. Measurements and Main Results: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. Conclusions: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.

Adipose-derived stem cells (ASCs) are readily available from autologous adipose tissue and have been demonstrated to provide significant potential for tissue rescue from, or repair of, damage in multiple animal models. These include models of myocardial infarction, heart failure, hind limb ischemia, and inflammatory conditions. Early clinical studies have now extended testing of the effects of ASC into patients. This review highlights some of the key reports underlining the potential of ASCs, focusing particularly on diseases involving the cardiovascular system, vascular growth, and tissue repair.Clinical applications of ASCs have begun to show early safety results and promising possibility of efficacy in patients with a range of diseases, including acute myocardial infarction, peripheral vascular disease, and soft and bony tissue defects including cranial bone loss, Crohn's-related fistula, and skin wounds. These effects are importantly based on the secretion of trophic and survival factors by these cells and by their participations in the growth and remodeling of blood vessels. These results suggest that ASCs could be a valuable therapeutic option in vascular growth and tissue repair in various clinical settings.ASCs may ultimately represent a valuable therapeutic option in tissue rescue and repair based on their ready availability, proangiogenesis and antiapoptotic factor secretion, immunomodulatory effects, and capacity for multilineage differentiation and ready expansion.

Adipose stromal cells (ASC) are multipotential mesenchymal progenitor cells that are readily induced to undergo adipogenic differentiation, and we have recently demonstrated them to have functional and phenotypic overlap with pericytes lining microvessels in adipose tissues. In this study we addressed the hypothesis that modulation of ASC fate within this perivascular niche can occur via interaction with endothelial cells (EC), which serve to modulate the adipogenic potential of ASC. To this end, we investigated contact as well as paracrine effects of EC on ASC adipogenesis, in two-dimensional coculture and via conditioned medium and analyzed mutual gene expression changes by real-time reverse transcription polymerase chain reaction (PCR). A significant decrease in adipogenic differentiation was observed in ASC when they were cocultured with EC but not control fibroblasts. This endothelial cell-specific effect was accompanied by increased expression of factors involved in Wnt signaling, most prominently Wnt1, Wnt4, and Wnt10a, which are well-known inhibitors of adipogenesis. Suppression of Wnt1 but not Wnt 10a or scrambled control short interfering RNA in cocultures partially reversed the endothelial cell effect, thus increasing adipogenic differentiation, suggesting a plausible role of Wnt1 ligand in modulation of adipogenesis by the vasculature. Furthermore, addition of recombinant Wnt ligand or the Wnt signaling agonist inhibited adipogenic differentiation of ASC in the absence of EC. In conclusion, these data define the relationship in adipose tissue between ASC and EC in the perivascular niche, in which the latter act to repress adipogenesis, thereby stabilizing vasculature. It is tempting to speculate that abnormal endothelial function may be associated with pathologic derepression of adipogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

The biological features of adipose stromal (stem) cells (ASC), which serve as progenitors for differentiated cells of white adipose tissue (WAT), are still largely undefined. In an initiative to identify functional ASC surface receptors, we screened a combinatorial library for peptide ligands binding to patient-derived ASC. We demonstrate that both primary and cultured human and mouse stromal cells express a conserved receptor targeted by peptides found to mimic SPARC, a matricellular protein that is required for normal WAT development. A signaling receptor for SPARC has not as yet been determined. By using the SPARC-mimicking peptides CMLAGWIPC (termed hPep) and CWLGEWLGC (termed mPep), isolated by panning on human and mouse cells, respectively, we identified the α5β1 integrin complex as a candidate receptor for SPARC. On the basis of these results, we evaluated ASC responses to SPARC or SPARC-mimicking peptide exposure. Our results suggest that extracellular SPARC binds to α5β1 integrin at sites of focal adhesions, an interaction disrupting firm attachment of ASC to extracellular matrix. We propose that SPARC-mediated mobilization of ASC through its effect on α5β1 integrin complex provides a functional basis for the regulation of WAT body composition by SPARC. We also show that α5β1 integrin is a potential target for ASC-selective intracellular delivery of bioactive peptides and gene therapy vectors directed by the SPARC-mimicking peptides. Disclosure of potential conflicts of interest is found at the end of this article.

It has been shown that stromal–vascular fraction isolated from adipose tissues contains an abundance of CD34 + cells. Histological analysis of adipose tissue revealed that CD34 + cells are widely distributed among adipocytes and are predominantly associated with vascular structures. The majority of CD34 + cells from freshly isolated stromal–vascular fraction were CD31 − /CD144 − and could be separated from a distinct population of CD34 + /CD31 + /CD144 + (endothelial) cells by differential attachment on uncoated plastic. The localization of CD34 + cells within adipose tissue suggested that the nonendothelial population of these cells occupied a pericytic position. Analysis of surface and intracellular markers of the freshly isolated CD34 + /CD31 − /CD144 − adipose-derived stromal cells (ASCs) showed that >90% coexpress mesenchymal (CD10, CD13, and CD90), pericytic (chondroitin sulfate proteoglycan, CD140a, and CD140b), and smooth muscle (α-actin, caldesmon, and calponin) markers. ASCs demonstrated polygonal self-assembly on Matrigel, as did human microvascular endothelial cells. Coculture of ASCs with human microvascular endothelial cells on Matrigel led to cooperative network assembly, with enhanced stability of endothelial networks and preferential localization of ASCs on the abluminal side of cords. Bidirectional paracrine interaction between these cells was supported by identification of angiogenic factors (vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor), inflammatory factors (interleukin-6 and -8 and monocyte chemoattractant protein-1 and -2), and mobilization factors (macrophage colony-stimulating factor and granulocyte/macrophage colony-stimulating factor) in media conditioned by CD34 + ASCs, as well a robust mitogenic response of ASCs to basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor-BB, factors produced by endothelial cells. These results demonstrate for the first time that the majority of adipose-derived adherent CD34 + cells are resident pericytes that play a role in vascular stabilization by mutual structural and functional interaction with endothelial cells.

Introduction Both Endothelial progenitor cells (EPC) and adipose stromal cells (ASC) are under investigation as therapies for cardiovascular diseases. Both cell types are capable of modulating vascular assembly and are, thereby, capable of directly promoting revascularization of ischemic tissues. We have shown that EPC differentiate into endothelial cells to form small vessels, whereas ASC have pericytic properties and naturally stabilize vessels. In this study we tested the possibility that ASC would interact with EPC to assemble de novo vessels in collagen in an in vivo chimeric implant. Methods and Results Collagen implants embedded with either umbilical cord blood EPC or adult ASC or a 4:1 mixture of both (2x10 6 cells/ml) were implanted subcutaneously into NOD/SCID mice. After 14 d implants were harvested and evaluated by immunohistochemistry. There was a pronounced difference among the groups in vascular network assembly. The majority of vessels formed in the EPC and ASC monocultures were small capillaries bounded by a single endothelial layer. Conversely, 100% of the plugs embedded with both cell types were highly invaded with multilayered arteriolar vessels. The density of the CD31 + vessels in the EPC and co-culture plugs was 26.6 ± 5.8 and 122.4 ± 9.8 per mm 2 , respectively. No CD31 + cells of human origin were detected in the ASC monocultures, indicating that ASC, which do not express this EC-specific marker, engage murine EC or form pseudovessels in this system. The density of α-SMA + vessels with lumens per mm 2 was 13.1 ± 3.6 (EPC), 10.2 ± 3.5 (ASC) and 124.7 ± 19.7 (co-culture). The total overlap of CD31 + and SMA + vessels demonstrates that mature, multilayered conduits were formed with the co-culture. Moreover, the majority of these vessels were filled with erythrocytes (92.5 ± 16.2 per mm 2 ), indicating inosculation with the native vasculature, which was confirmed by ultrasound with echogenic microbubbles and persisted to at least 4 months. Conclusion This study is the first to demonstrate that non-transformed human EPC and ASC cooperatively form mature and stable vasculature with subsequent functional integration into a host vasculature system.

The use of adipose-derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual-cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC-shHGF and ASC-shCtrl, respectively). Transduced ASC-shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC-shHGF were also significantly impaired, compared with ASC-shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP+ cells were detected at 3 weeks in ischemic limbs of mice treated with ASC-shHGF compared with those treated with ASC-shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.

Urokinase plasminogen activator (uPA) is required for both endogenous and vascular endothelial growth factor (VEGF)-augmented angiogenesis in normal tissues, leading us to hypothesize that uPA augmentation by gene transfer might promote angiogenesis in ischemic tissues. Overexpression of uPA was studied in rat myocardial infarction (MI) and mouse hind limb ischemia models and compared with VEGF overexpression effects. Animals were divided into control and three experimental groups (n = 6), receiving intramuscular injections of plasmids as follows: (i) control (empty vector or expressing beta-galactosidase); (ii) uPA; (iii) VEGF(165); (iv) a 1:1 mixture of uPA and VEGF(165). The capillary densities in both ischemic models were greater (P0.05) in tissues treated with uPA, VEGF, or a combination of both than in controls. Infarct size was reduced in hearts from uPA and VEGF experimental groups compared with controls (P0.05). Local overexpression of uPA induced a marked increase in the number of macrophages and myofibroblasts present within infarcts. Hind limb blood flow was greater in all experimental groups by day 10 (P0.05). Overall, the effects of uPA and VEGF were uniformly comparable. Additional analysis revealed association of local edema with VEGF but not with uPA treatment. This study established that uPA gene therapy effectively induces functionally significant angiogenesis in models of acute MI and hind limb ischemia.