ADIPOSE-DERIVED STROMAL CELLS PROMOTE SURVIVAL OF ENDOTHELIAL CELLS AND KERATINOCYTES IN WOUND HEALING MODEL

poster abstractBurn wounds are a significant medical challenge today. Current treat-ment includes the use of skin grafts or wound healing scaffolds to protect the wound and promote healing. However, pre-existing conditions and fac-tors such as smoking can compromise normal healing thru decreased growth factor production, prolonged inflammation, tissue hypoxia, reduced cellular migration and ECM deposition, and impaired revascularization, making the wound more susceptible to infection. Adult pluripotent cells have been proposed as a therapy for multiple dis-orders because they have been shown to decrease inflammation and pro-mote host tissue preservation and angiogenesis. Adipose-derived stromal cells (ASC) are a population of mesenchymal, pluripotent cells derived from adipose tissue. Compared to bone marrow derived MSC, ASC can be easily obtained thru minimally invasive procedures. It has been shown in previous studies that ASC improved wound closure in normal and diabetic mice and stimulated proliferation of human dermal fibroblasts, increasing the epitheli-alization of cutaneous wounds. The next challenge is to find a clinically relevant cell-delivery method. In light of this, we propose the use of current clinical wound healing scaf-folds as a delivery vehicle for ASC in combination with endothelial cell (EC) and keratinocytes. We hypothesize that that ASC will promote keratinocyte and EC survival (both are used clinically), thus promoting epithelialization and neovascularization of graft. The use of ASC, EPC and keratinocytes in combination with wound healing scaffolds currently used by physicians, such as Integra is a novel combination and will provide a faster transition to clinic if it is found to be efficacious. Our lab has shown that ASC promote survival of EC on Integra and sup-port the formation of vascular-like cord structures. Factors secreted by ASC promote keratinocytes ingrowth in a wound closure assay. Keratinocytes also showed increased survival when cultured with ASC

Human adipose stromal cell therapy improves survival and reduces renal inflammation and capillary rarefaction in acute kidney injury

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

Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium

Stromal fibroblasts modify the initial recruitment of leucocytes by endothelial cells (EC), but their effects on subsequent transendothelial migration remain unclear. Here, EC and dermal or synovial fibroblasts were cultured on opposite surfaces of 3-lm pore filters and incorporated in static or flow-based migration assays. Fibroblasts had little effect on tumour necrosis factor-a-induced transendothelial migration of neutrophils,but tended to increase the efficiency of migration away from the endothelium.Surprisingly, similar close contact between EC and fibroblasts strongly reduced lymphocyte migration in static assays, and nearly abolished stable lymphocyte adhesion from flow. Fibroblasts did not alter endothelial surface expression of adhesion molecules or messenger RNA for chemokines. Inhibition of attachment did not occur when EC-fibroblast contact was restricted by using 04-lm pore filters, but under these conditions pre-treatment with heparinase partially inhibited adhesion. In the 3-lm pore co-cultures, inhibition of metalloproteinase activity partially recovered lymphocyte adhesion, but addition of CXCL12 (SDF-1a) to the endothelial surface did not. Hence, the ability of EC to present activating chemokines for lymphocytes may have been enzymatically inhibited by direct contact with fibroblasts. To avoid contact, we cultured EC and fibroblasts on separate 3-lm pore filters one above the other. Here,fibroblasts promoted the transendothelial migration of lymphocytes. Fibroblasts generate CXCL12, but blockade of CXCL12 receptor had no effect on lymphocyte migration. While stromal cells can provide signal(s)promoting leucocyte migration away from the sub-endothelial space,direct cell contact (which might occur in damaged tissue) may cause disruption of chemokine signalling, specifically inhibiting lymphocyte rather than neutrophil recruitment

Human adipose derived stromal/stem cells (hASCs) protect against STZ-induced hyperglycemia; analysis of hASC-derived paracrine effectors

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

Intravenous xenogeneic transplantation of human adipose-derived stem cells improves left ventricular function and microvascular integrity in swine myocardial infarction model

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 × 10(7) ASCs), or the divided dose group (DD) (n = 7, 5 × 10(7) 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

Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia

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

Vascular and Cardiac Adult Stem Cell Therapy Center

poster abstractThe mission of the Vascular and Cardiac Adult Stem Cell Therapy Center (VC-CAST) is the discovery and clinical translation of therapies involving transplantation of adult stem cells into patients with debilitating diseases. To accomplish this, VC-CAST fosters multidisciplinary research collaborations that address both biology of adult stem cells that are readily available, and the translation of their study from the laboratory into clinical trials. The use of such cells is highly feasible, and not ethically controversial, as they are derived from readily-available tissues such as fat and bone marrow. Since its inception, VC-CAST projects have been multidisciplinary, involving multiple clinical as well as basic departments of the School of Medicine. VC-CAST projects are also collaborative, with most of the projects having one or more industrial partners. A key partnership has also been established by the creation of the Veterans Affairs Center for Regenerative Medicine (VACRM) at the Roudebush VA Medical Center in Indianapolis, which will provide a unique referral site focusing on research and implementation of first-in-human trials in the fields of poor circulation, arthritis, wound healing, diabetes, and emphysema. Given the focus of VC-CAST researchers on translation, the center is active in pursuit of intellectual property that is critical to building corporate engagement and thus the enablement of translation to clinical trials. Signature center funding has allowed IUPUI investigators to try high-risk, high-reward ideas, which could not otherwise be funded readily, via either NIH or venture-capital methods. Most of these experiments are still ongoing, but have already led to discoveries of potentially critical significance to patients. The novelty of some of these discoveries promises to attract new funding, as well as to provide bases for potential licensing revenues and startup opportunities. This poster will highlight several of these projects, representative of center activities in their collaborative, multidisciplinary and translational and potentially commercializable aspects. Some key projects are as follows: • Based on recent completion of the Phase I/II clinical trial, “Stem cell Angiogenesis to promote limb salVagE (SAVE), a new randomized Phase III clinical trial testing the use of one’s own bone marrow-derived stem cells to save legs from amputation has been initiated, with Dr. Murphy as the national PI. • Adipose Stem Cells for Peripheral Arterial Disease. • Endometrial Regenerative Cells for Peripheral Arterial Disease. • Adipose Stem Cells for treatment of Heart Attack and prevention of Heart Failure. • Adipose Stem Cells for Emphysema and other Lung Diseases • Adipose Stem Cells for Prevention and Treatment of Diabetes • Isolation and Characterization of Endothelial and Mesenchymal Stem Cells from Term Human Placenta. • Isolation and Characterization of Endothelial Colony Forming Cells (ECFCs) from Human Adult Blood Vessel

Assessing the Permeability of Engineered Capillary Networks in a 3D Culture

Many pathologies are characterized by poor blood vessel growth and reduced nutrient delivery to the surrounding tissue, introducing a need for tissue engineered blood vessels. Our lab has developed a 3D co-culture method to grow interconnected networks of pericyte-invested capillaries, which can anastamose with host vasculature following implantation to restore blood flow to ischemic tissues. However, if the engineered vessels contain endothelial cells (ECs) that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this study was to test the resistance to permeability of these networks in vitro, grown with different stromal cell types, as a metric of vessel functionality. A fluorescent dextran tracer was used to visualize transport across the endothelium and the pixel intensity was quantified using a customized MATLAB algorithm. In fibroblast-EC co-cultures, the dextran tracer easily penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, dextran accumulated at the periphery of the vessel, with very little transported across the endothelium. Quantitatively, permeability dropped from initial levels of 61% to 39% after 7 days, and to 7% after 2 weeks. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), much tighter control of permeability was achieved. Relative to the EC-fibroblast co-cultures, permeabilities were reduced 41% for the EC-MSC co-cultures and 50% for the EC-AdSC co-cultures after 3 days of culture. By day 14, these permeabilities decreased by 68% and 77% over the EC-fibroblast cultures. Co-cultures containing stem cells exhibit elevated VE-cadherin levels and more prominent EC-EC junctional complexes when compared to cultures containing fibroblasts. These data suggest the stromal cell identity influences the functionality and physiologic relevance of engineered capillary networks