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IFATS 09 - Top Abstracts

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Methods:
The APOLLO is a prospective, double-blind, randomized, placebo-controlled, dose escalation cell therapy study that will enroll up to 48 patients to determine the safety and feasibility of different doses of adipose-derived stem cells delivered via the intracoronary route in the treatment of patients with ST-elevation myocardial infarction.

Results:
The study includes up to four consecutively enrolled dose cohorts of 12 patients each. Within each dose cohort, patients are randomly assigned 3:1 to receive cell therapy or placebo. Eligible patients undergo liposuction and intracoronary infusion of the adipose-derived regenerative cells isolated from the lipoaspirate within 24 hours after the primary PCI. Patients are evaluated for 36 months, whereas 3D echocardiography, SPECT scintigraphy, invasive hemodynamic analysis (P/V loop analysis), cardiac MRI and BNP blood analysis at 6 and 18 months are used to evaluate global and regional cardiac function and perfusion. Currently, 7 patients have been enrolled in the first dose cohort, whereas the therapy has been deemed safe in these patients.

Conclusions:
The APOLLO study is a prospective, does-escalating cell therapy study to evaluate the safety and feasibility of adipose-derived regenerative cells in the treatment of patients with a myocardial infarction.

 

 


Evidence of a robust trafficking capability: Adipose-derived stem cells slowly roll on P-selectin and enhance firm adhesion to VCAM-1 following exposure to SDF-1alpha

Alexander Bailey*; Michael B. Lawrence*; Hulan Shang**; Adam Katz**; Shayn Peirce*

*Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA
** Department of Plastic Surgery, University of Virginia, Charlottesville, Virginia, USA

Introduction:
Intravenous delivery of adipose-derived stem cells (ASCs) is a promising option for the treatment of ischemia. Following delivery, ASCs that reside and persist in the injured extra-vascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limits the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic ASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting critical adhesion proteins. Towards this end, we investigated cellular- and molecular-level interactions between ASCs and endothelial cells under flow conditions, in vivo and in vitro, to identify methods to increase the number of cells that successfully traffic to and incorporate into injured/ischemic tissues.

Methods:
Early passage human hASCs (P=0-3) were perfused at a wall shear stress equal to 0.5 dynes/cm(2) over immobilized human VCAM-1 or P-selectin in a parallel plate flow chamber. Firm adhesion and slow rolling interactions with these endothelial cell adhesion molecules were recorded and quantified. To identify which hASC adhesion molecules may be mediating these interactions, hASCs were incubated with competitive antibodies to PSGL-1 or alpha4 integrins for 30 minutes prior to assay, and flow cytometry was preformed separately. For in vivo studies, DiI-labeled hASCs (P=3; 750,000 cells/ml) were injected into the mesenteric artery of male rats, and the subsequent adhesive interactions between injected cells and exposed mesenteric microvascular endothelium were observed in real time using intravital microscopy. hASCs were also chemically pre-conditioned to increase adhesive interactions by incubating with SDF-1alpha (0.1 microg/ml) for 30 minutes prior to both the in vitro and in vivo assays.

Results:
Under flow conditions, hASCs slowly rolled on VCAM-1 and P-selectin at speeds as low as 2 microns/sec and exhibited the characteristic stop-and-go behavior typical of rolling leukocytes. Furthermore, hASCs firmly adhered to VCAM-1 (8-cells/mm (2) min), which was enhanced following SDF-1alpha pre-conditioning (23-cells/mm(2) min). Incubation of hASCs with competitive antibodies to the alpha4 integrins eliminated all VCAM-1 interactions, while blocking PSGL-1 activity had no effect on hASC ability to roll on P-selectin. These data were complemented by in vivo observations that hASCs slowly rolled and firmly adhered to injured endothelium in patent arterioles and venules in rat mesenteric microvasculature. Similar to in vitro studies, a greater number of SDF-1alpha pre-conditioned hASCs (versus non-conditioned hASCs) persisted in the microvasculature (8.0% versus 1.3%) at 1 hour following injection.

Conclusions:

Our study suggests that hASCs possess robust trafficking capabilities and interact directly with injured endothelium by capitalizing on well-known molecular adhesion interactions. Moreover, we present a viable method for enhancing these interactions using a biochemical pre-conditioning scheme, with the long term goal of improving hASC trafficking efficiencies.

 

 


Regulation of lymphocyte proliferation by human adipose-derived stem cells requires
IFN-gamma, IDO activity and generates T cells with suppressor activity

Olga De La Rosa*

* Medical Oncology, Department of Internal Medicine, University Hospital Zurich, Zurich, Switzerland

Introduction:
Human adipose-derived stem cells (hASCs) are mesenchymal stem cells with reduced immunogenicity and the capability to modulate immune responses. These properties make hASCs of special interest as therapeutic agents in the settings of both chronic inflammatory and autoimmune diseases. Although similar to bone-marrow mesenchymal stem cells (BM-MSC), hASCs may differ from them in the mechanism of immunosuppression. We have studied the mechanism responsible for the hASC-mediated immune regulatory effect.

Methods:
hASCs isolation and expansion. Lymphocyte proliferation assays. CD3+, CD4+, CD8+ isolation. Cytometric bead arrays. Flow cytometry. Neutralization assays. HPLC.

Results:
We report that hASCs inhibit PBMC, CD4+ and CD8+ T cell proliferation in both cell-cell contact and transwell conditions. This inhibition is accompanied by a reduction of proinflammatory cytokines. We demonstrate that hASCs do not constitutively express immunomodulatory factors. Conditioned supernatants from hASC stimulated by IFN, PBMCs or activated PBMCs highly inhibited PBMC proliferation, indicating that inhibitory factors are released upon hASC activation. hASC-mediated immunosuppression is highly mediated by IFN and IDO activity. Furthermore, interaction between hASCs and PBMCs generates CD3+ T cells with a regulatory phenotype.

Conclusions:
hASCs prevent lymphocyte proliferation by releasing soluble factors upon activation through inflammatory mediators and contribute to the generation of regulatory T cells. Collectively, these data provide immunological support for the safety and efficacy of hASCs in the treatment of inflammatory diseases.

 

 


Safety and efficacy of autologous adipose-derived stromal cells on Type II diabetes patients: 6 month post-procedure results

Florencio Lucero*; Emerita Barrenechea**; Letitia Lucero-Palma*; Kribah Krishnan*; Eduardo Barrenechea; Bill Paspaliaris*

* AdiStem Ltd, Hong Kong
**Veterans Memorial Hospital, Manila

Introduction:
Stem cell therapies hold great promise for anti-aging benefits as they are regenerative in nature. Autologous adipose-derived stem cell transplants hold even more potential as they have no ethical barriers and require no out-of-surgery culture requirements.

Methods:
We have devised a procedure that entails the isolation of stromal cells from adipose-tissue derived from a mini-liposuction procedure, their activation from a quiescent stage to an active stage, and their reintroduction back into the patient via intravenous mode. This single procedure has now been performed on 176 subjects over a two and a half year period in four countries with no adverse effect. Because these were isolated case studies, a formal clinical trial was then initiated to assess the safety and efficacy of the procedure on a controlled group of 34 patients with non-insulin and insulin-dependent type II diabetes mellitus with no cardiovascular or nephrological complications.

Results:
After three months post-operation the patients showed a significant and sustained reduction in fasting glucose levels (from 9.64+3.88 mmol/l to 7.01+1.64 mmol/l; p=0.005 at 2 weeks to 7.71+2.29 mmol/l; p=0.01 at 12 weeks), glycosylated haemoglobin (from 9.11+2.06% to 7.73+1.19%;p=0.00001), C-peptide (from 2.75+1.02 to 2.27+1.45; p=0.045) and triglycerides (from 2.31+1.53 to 1.91+1.63;p=0.03). At six months post procedure nearly half the patients reverted back to pre-op conditions while the other half continues to see sustained decreases in diabetic parameters as compared to pre-op levels. Six month statistics showed Fasting blood sugar went from 9.64+3.88 mmol/l at pre-op to 8.50+2.86 mmol/l; p=ns at 24 weeks), glycosylated haemoglobin (from 9.11+2.06% to 8.10+1.82%;p=0.001), C-peptide (from 2.75+1.02 to 2.83+1.37; p=ns) and triglycerides (from 2.31+1.53 to 2.01+1.35;p=ns). Most patients have noticed an increase in well-being parameters post-op. There was no significant change detected post-op in total cholesterols and other CBC, LFT and KFT values and no obvious adverse reaction has been noted.

Conclusions:
The results of the trial to date suggest that the autologous adipose derived stromal cell therapy appears to be safe and beneficial to type II diabetes patients by decreasing their resistance to insulin and decreasing diabetic cardiovascular risk factors. We believe that the stromal cell transplant is probably acting by increasing adiponectin levels in these subjects, an adipocytokine that is produced by adipose stromal cells and known to regulate insulin--resistance. Lifestyle and hypoglycemic medication changes also plays an important role in sustaining the effects observed.

 

 


Immunogenicity of Allogeneic Adipose-Derived Stem Cells in a Rat Spinal Fusion Model

Nakia Spencer*; Paul Anderson**; Jeffrey Gimble***; Kevin McIntosh****; Mandi Lopez*; Jade Borneman****

*Laboratory for Equine and Comparative Orthopedic Research, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Skip Bertman Drive, Louisiana State University, Baton Rouge, Louisiana 70803
**Department of Orthopedics & Rehabilitation, University of Wisconsin-Madison, Madison, Wisconsin 53792
*** Stem Cell Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana 70808
****Cognate BioServices, Inc., 1448 S. Rolling Road, Baltimore, Maryland 21227

Introduction:
Adipose-derived stem cells (ASCs) express a non-immunogenic profile as shown by in vitro studies which demonstrate a lack of T cell proliferation to allogeneic ASCs as well as ASC mediated suppression of mixed lymphocyte reactions (MLR). To determine whether these observations translate in vivo, immune monitoring studies were carried out in rats transplanted with allogeneic ASCs as part of a scaffold-based spinal fusion study.

Methods:
A total of 56 Fischer strain rats were randomly assigned to four different treatment cohorts after bilateral decortication of the L4 and L5 transverse processes (n=14/cohort). ASCs, derived from Fischer or ACI rats, were loaded onto scaffolds and implanted in Fischer recipients according to the following treatments: 1) No treatment; 2) Scaffold only; 3) Syngeneic ASCs + Scaffold; or 4) Allogeneic ASCs + Scaffold. Half of each group was sacrificed at 4 weeks post implantation and the remaining animals were sacrificed at 8 weeks. Cellular and humoral immune responses were evaluated at both timepoints to determine whether donor ACI strain ASCs induced an immune response in recipient Fischer rats. Recipient Fischer lymph node cells were assessed for T cell proliferation to ACI spleen cells by one-way MLR assays and serum was collected to assess antibody binding to donor ASCs by flow cytometry.

Results:
No T cell priming to donor ACI alloantigens was observed in recipients at 4 or 8 weeks after implantation of ASCs as determined by the kinetics and magnitude of MLR responses. A significant antibody response was detected in Fischer recipients implanted with either Fischer or ACI strain ASCs when compared to the "No Treatment" group. Antibodies were of the IgG isotype and non-cytotoxic in the presence of complement. Antibodies specific for fetal bovine serum proteins were detected in the "Scaffold Only", “Syngeneic ASCs + Scaffold” and “Allogeneic ASCs + Scaffold” groups.

Conclusions:
These results support the use of allogeneic ASCs for spinal fusion and suggest that ASCs be propagated in syngeneic serum to mitigate the production of antibodies.

 

 


Anti-adipogenic effect of adipose tissue T lymphocytes on the human CD34+/CD31- cells

Carine Duffaut*; Alexia Girard*; Coralie Sengenes*; Jean Galitzky*; Anne Bouloumié*

* Institut National de la Santé et de la Recherche Médicale, U858, AVENIR Team, I2MR, Paul Sabatier University, IFR31, Toulouse, France

Introduction:
Obesity is characterized by a low grade inflammatory state. The white adipose tissue appears as a primary player in the settlement of such a condition as both source and site of inflammation. Accumulation of macrophages within the fat mass in obese conditions has been well described but the potential role of their major cell partners, i.e. T-lymphocytes, has been less studied. The aim of the present study was to describe the population of human adipose tissue lymphocyte (ATLs) and to study their effects on adipogenesis.

Methods:
Flow cytometry analyses were performed on the stroma-vascular fraction (SVF) of human subcutaneous adipose tissue of lean to obese patients (n=130) using cell surface markers of lymphocyte subtypes (CD3, CD19, CD4, CD8, CD56, CD45RO, CD45RA). Human adipose tissue progenitor cells (CD34+/CD31-) and T-lymphocytes (CD3+) were isolated through immunoselection/depletion protocols from the human SVF. CD3+ transcript expression was analysed by real time PCR analysis. Conditioned media from native human ATLs, obtained after 24h culture period of CD3+ cells, were used to treat CD34+/CD31- cells maintained under adipogenic culture conditions.

Results:
Flow cytometry analyses demonstrated the presence of T-lymphocytes (CD3+) in human subcutaneous adipose tissue that are increased with the body mass index of the patients. ATLs were mainly composed by memory (CD45RO+/CD45RA-)and effector (CD45RO-/CD45RA-) helper (CD4+) and cytotoxic (CD8+) T cells. Analysis of the ATLs transcript expression showed high amount of RANTES, interferon gamma as well as TNFalpha Treatment of the native CD34+/CD31- cells cultured under adipogenic culture conditions with ATL-derived conditioned media led to a strong inhibition of cell number associated with decreased adipogenesis assessed by triglyceride accumulation and expression of adipocyte specific markers lipoprotein lipase and fatty acid synthase.

Conclusions:
The present study demonstrates that obesity in human is associated with accumulation of T-lymphocytes, i.e. helper as well as cytotoxic T cells, within the adipose tissue. Such a cell population might play a major role through their anti-adipogenic effect on the extension capacity of the fat mass and thus indirectly to the obesity-associated pathologies

 

 


Endothelial differentiation of adipose-derived stem cells: How close have we really come?

Paul DiMuzio*; Ping Zhang*; Stephen McIlhenny*; Lisa Harris*; Hamid Abdollahi*; Matthew Ferroni*; Kristi Wasson*; Eric Hager*; Neil Moudgil*; Nicholas Tarola*; Daniel Grabo*; Vic Srinivas*; Irving Shapiro*; Thomas Tulenko*

*Department of Surgery, Thomas Jefferson University, Philadelphia, PA

Introduction:
We have demonstrated that adipose-derived stem cells (ASC) are abundant in the peri-umbilical fat of patients with cardiovascular disease, suggesting these cells may be practical for use in tissue engineering/regenerative medicine. In particular, we have focused on their use in creating a tissue engineered vascular graft produced by seeding ASC differentiated into endothelial cells (EC) onto a natural vascular tissue scaffold. The current studies evaluate the efficacy of growth factors, shear stress, and hypoxia in stimulating ASC to acquire EC characteristics.

Methods:
ASC (CD13+29+90+31-45-) isolated from the peri-umbilical fat of patients undergoing vascular surgery were cultured in either Endothelial Cell Growth Supplement (ECGS) or EGM2 media (up to 4wk), under normoxic vs. hypoxic (21% vs. 2% O2, up to 2wk) conditions, with or without the application of shear stress (12dyne x 72h) in vitro. After differentiation, we assessed: 1) expression of eNOS, vWF, CD31, veCadherin, VEGF, 2) cord formation in Matrigel, 3) re-alignment in the direction of shear force, and 4) production of nitric oxide (NO). The effect of hypoxia was determined by expression of HIF1α. In a canine model, differentiated ASC were seeded onto vascular grafts and implanted within the carotid arterial circulation for two weeks. Explanted grafts underwent histological evaluation to assess endothelial function of the stem cells in vivo.

Results:
Culture in either ECGS or EGM2 for 1 wk resulted in ability to form cords and re-align in the direction of shear. EGM2 stimulated more vWF and CD31 expression than ECGS at 2wk, but significantly less than in EC controls. In cultures differentiated for 2wk, the addition of shear stress increased both vWF and CD31 expression. The addition of hypoxia (and resultant HIF1α upregulation), however, decreased CD31 expression, despite significant increases in VEGF expression. None of the stimuli resulted in expression of eNOS or veCadherin; only the addition of VEGF (50ng/ml) to EGM2 medium resulted in minimally detectable levels of eNOS at 3wk. NO was detected in the media of cells cultured in either ECGS and EGM2 for 2wk, but at levels inferior to control EC; the differentiated stem cells expressed nNOS, but not eNOS or iNOS. Finally, at physiological levels of shear, only ECGS-cultured cells remained attached to vascular grafts. After 2wks in vivo, 6 of 7 ECGS-cultured stem cell grafts remained patent; however, the luminal surfaces of the patent grafts were uniformly coated with fibrin.

Conclusions:
These results suggest that adipose-derived stem cells acquire several characteristics associated with endothelial cells when stimulated by various growth factors and shear stress. Conversely, hypoxic conditions appear to promote their “stemness.” Despite these manipulations of the microenvironment in vitro, the stem cells do not express significant amounts of eNOS and appear mildly thrombogenic in vivo, two essential characteristics of differentiated endothelial cells. We therefore conclude that the ability of these tissue-specific stem cells to commit fully to an endothelial lineage is limited, and that genetic manipulation is likely required to bring about full differentiation.

 

 


Therapeutic effect of adipose-derived mesenchymal stem cells on experimental colitis by inhibiting inflammatory and autoimmune responses

Laura Rico*

*Cellerix SL, Spain

Introduction:
Crohn’s disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon partially caused by a loss of immune tolerance against mucosal antigens. Mesenchymal stem cells (MSCs) of allogeneic origin have been reported to suppress effector T-cell responses in vitro, to have therapeutic effects in some immune disorders and certain capacity to restore immune tolerance. The aim of this work is to investigate the potential anti-inflammatory and therapeutic effect of human adipose-derived MSCs (hASCs) in two well-established murine models of inflammatory bowel diseases.

Methods:
We examined the therapeutic action of hASCs in the colitis-induced by administration of trinitrobenzene sulfonic acid or dextran sodium sulfate, evaluating diverse clinical signs of the disease. We also investigated the mechanisms involved in the potential therapeutic effect of hASCs, such as inflammatory cytokines and chemokines, Th1-type response, and the generation of regulatory T (Tr) cells.

Results:
Systemic infusion of hASCs significantly ameliorated the clinical and histopathologic severity of colitis, abrogating body weight loss, diarrhea, and inflammation, and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response, by regulating a wide spectrum of inflammatory mediators directly through activated macrophages, and by generating interleukin-10-secreting Tr cells with suppressive capacity on autoreactive T cells. In addition, hASCs protected from mortality caused by sepsis by downregulating the exacerbated inflammatory response characteristic of this disease.

Conclusions:
Therefore, these adult MSCs emerge as key regulators of immune tolerance in physiological conditions by inducing the generation/activation of Tr clones in the periphery and as attractive candidates for a cell-based therapy for the treatment of inflammatory and autoimmune disorders.

 


Abstract ID: 88

In vivo effects of hypoxia on adipose tissue: Implications for obesity-related dysfunction

Hirotaka Suga; Hitomi Eto; Kotaro Yoshimura

Introduction:
It has been suggested that hypoxia and chronic inflammation of adipose tissue induced by obesity are associated with dysfunction of adipose tissue, leading to insulin resistance and metabolic syndrome. Hypoxia has been known to have various effects on adipocytes and adipose-derived stromal cells (ASCs), but direct effects of hypoxia independent from obesity have been examined only in vitro, presumably because there is no good model for non-obese hypoxic adipose tissue.

Methods:
We established a new animal model for hypoxic adipose tissue in mice by transecting nutrient vessels for the inguinal fat pad. We prepared three types of hypoxia models; mild, intermediate, and severe hypoxia. Interstitial partial pressure of oxygen (pO2) was measured with a needle-type oxygen sensor. Using a real-time RT-PCR, we examined mRNA expression of various genes, which have been reported to change in obesity. Histological changes and cellular events after hypoxia were examined with immunohistology of sectioned and whole mount tissues.

Results:
Immediately after hypoxia induction, mild-hypoxia, intermediate-hypoxia, and severe-hypoxia models showed pO2 of 37.3 mmHg, 29.2 mmHg, 11.6 mmHg, respectively, while sham models showed pO2 of 50.5 mmHg. In every model except sham, HIF1a, IL-1b, IL-6, TNFa, PAI-1, and GLUT1 were up-regulated as early as at 6 hours, while HGF and MMP2 were up-regulated as late as on day 7. However, the upregulations of these genes were temporary. On the other hand, leptin, adiponectin, and PPAR gamma were down-regulated as early as at 6 hours, and the down-regulation of these transcripts continued through 1 month and appeared to be irreversible. Only the severe-hypoxia model showed a prolonged hypoxic condition (pO2 of 27.0 mmHg at 2 weeks) and degenerative changes of adipose tissue frequently seen in obesity, while other models showed a gradual improvement of pO2 within 7 days and little degenerative changes in histology. The degenerative changes in the severe-hypoxia model resulted in atrophy (a lower weight of the adipose tissue) after 1 month. Under the severe hypoxia, Ki67+ proliferating cells increased on day 3, peaking on day 7. Many ASCs (CD34+/lectin–) were also detected on day, suggesting ASCs were involved in the remodeling process of the hypoxic adipose tissue. Aggregation of F4/80-positive macrophages was observed in the later phase, from day 14.

Conclusions:
Dysfunction of adipose tissue was seen under an experimentally-induced hypoxia. Although mild hypoxia (70% of normal pO2) temporarily induced the dysfunction, prolonged and severe (below 50% of normal pO2) hypoxia resulted in seemingly-irreversible dysfunction as well as degenerative changes in adipose tissue, which further induced adaptive and remodeling cellular events.

 


Abstract ID: 132

Transforming Growth Factor-β Is a Key Factor Responsible For Preventing ASC Adipogenesis and Promoting a Pericytic Phenotype

Dmitry Traktuev; Stephanie Merfeld-Clauss; Gangaraju Rajashekhar; Matthias Clauss; Keith March

Introduction:
We have previously shown that adipose stromal cells (ASCs) reside in vascular periendothelial layers where they may function as pericytes. Additionally, in vitro and in vivo experiments demonstrated that isolated ASCs readily undergo adipogenesis. The control mechanisms governing the switch among progenitor pericytic and differentiated adipocyte states of ASCs are not well defined. Based on the fact that ASCs in adipose tissue are in direct contact with endothelial cells (EC), we hypothesize that ECs, either through physical interaction or by paracrine signaling, are fundamentally important in controlling ASC transitions between these two states.

Methods:
ASC (passage 2-5) alone or in co-culture with ECs (passage 5) at a 1:1 ratio were exposed to (1) control basal medium (EBM-2/5%FBS), (2) adipogenic differentiation medium/5%FBS (ADM), or (3) ADM combined at a ratio of 1:2 with either basal or EC conditioned medium (EC-CM). EC-CM was produced by culturing EC in basal medium for 72h. Protein analysis of EC-CM was performed using an antibody array (RayBiotech). To evaluate the effects of TGFβ contained in EC-CM on ASC adipogenic potential, media were supplemented with neutralizing antibodies to TGFβ. To directly evaluate the effect of TGFβ on adipogenesis and smooth muscle cell differentiation, ASC were grown in ADM or basal medium with and without TGFβ supplementation. Adipocyte or smooth muscle cell formation was assessed, respectively, by Nile Red staining or immunological staining with -smooth muscle actin antibodies.

Results:
Co-culture of ASCs with ECs in ADM suppressed adipogenesis of ASCs. To determine if this effect was due to factors secreted by ECs, we exposed ASCs to the ADM:EC-CM medium. Culturing ASCs in ADM: EC-CM medium also significantly decreased adipogenesis compared to the cells exposed to ADM/EBM-2/5%FBS. Analysis of the EC-CM revealed that ECs secret TGFβ during culture. The presence of TGFβ in ADM significantly inhibited lipid accumulation in ASCs (up to 63%). Decreased expression of lipase and PPARγ mRNA expression was also observed. Pretreatment of EC-CM with anti-TGFβ significantly decreased the suppression of adipogenesis by EC-CM. Similar results were observed in co-culture experiments where the presence of anti-TGFβ in ADM enhanced ASC adipogenesis. Analysis of direct effects of TGFβ on ASCs revealed upregulation of α-smooth muscle actin expression and its organization into stress fibers. Conversely, pre-treatment of ASCs in ADM with TGFβ reduced adipogenesis.

Conclusions:
Based on the results of this study we conclude that the factors secreted by EC are involved in modulating differentiation of ASCs to adipocytes. The major factor involved in regulating this particular switch is TGFβ.

 


Abstract ID: 89

Involvement of adipose-derived stromal cells in remodeling and expansion process of adipose tissue

Hirotaka Suga; Hitomi Eto; Harunosuke Kato; Kotaro Yoshimura

Introduction:
Adipose-derived stromal cells (ASCs) are considered to act as tissue-specific progenitor cells and have multipotency, but actual cellular events in remodeling or expansion process of adipose tissue are not well studied. Unlike conventional histological analysis of adipose tissue, whole mount histology using a confocal microscopy-based method [Nishimura et al. Diabetes, 56: 1517, 2007] enables detailed and 3-dimensional visualization of adipose tissue such as relationships between adipocytes, other cellular components and vessels.

Methods:
We analyzed remodeling and expansion processes of adipose tissues using original murine models for ischemia-reperfusion and external tissue suspension, respectively. For whole mount staining of living adipose tissue, samples were stained with fluorescent agents (BODIPY for adipocytes, lectin for endothelial cells, and Hoechst 33342 for nuclei), and images (single, multiple-serial, or surface-rendered 3-dimensional images) were captured by a confocal microscopy. Other agents were also combined to further analyze cellular events; propidium iodide (PI) for necrotic cells acetylated low-density lipoprotein (acLDL) for endothelial progenitor cells, and anti-CD34 antibody for ASCs.

Results:
After ischemia-reperfusion, adipose tissue underwent a remodeling process (degeneration and regeneration of adipocytes and capillaries), while adipose tissue expansion (adipogenesis and angiogenesis) was seen during a continuous external tissue suspension. After ischemia-reperfusion injury, increase of interstitial space and small-sized adipocytes (smaller than 50 μm in diameter), an increased number of nucleated cells including lectin-positive round cells, and capillaries especially around small-sized adipocytes were observed. Combination with PI enabled detection of necrotic adipocytes, which appeared 3 and 7 days after injury.

We also detected migration of endothelial progenitor cells (acLDL+/lectin+), which were most frequently observed 7 days after injury. With an antibody against CD34, pericytic localization of ASCs and their proliferation and increase in number after injury were confirmed. During a continuous external tissue suspension, a rapid thickening of adipose tissue layer, increase in small-sized adipocytes and ASCs (CD34+/lectin–), and an increased density of capillaries and small vessels arranged in the direction of external suspension were observed. Most of Ki67+ proliferating cells were lectin-negative and suggested to be ASCs, and a substantial number of CD34+/lectin+ cells, which may be ASCs differentiating into endothelial cells, were seen especially in the marginal area of expanding adipose tissue on day 14.

Conclusions:
Three-dimensional visualization of adipose tissue with a confocal microscopy and fluorescent agents provided us with detailed images of adipose tissue, which helped us precisely examine cellular events in adipose tissue in various situations. ASCs were highly involved in adipose tissue remodeling and expansion as a main dividing cell population and contributed to adipogenesis and angiogenesis.

 


Abstract ID: 124

Direct co-culture of human adipocytes, adipose tissue macrophages and adipose stem/progenitor cells (ASPCs) reveals a mechanism for generation of new preadipocytes and a novel population of CD34 (+) ASPCs

Saleh Heneidi; Cristina Bertolotto; Gregorio Chazenbalk; Charles Simmons; Ricardo Azziz

Introduction:
Increased obesity is associated with accumulation of macrophages in adipose tissue. In addition to regular phagocytotic function, adipose tissue macrophages may also influence the adipocyte growth as well as its metabolism and secretory activity through the production of cytokines and chemokines. We have recently demonstrated that CD14(+) adipose tissue macrophages represent a potential progenitor pool for preadipocytes through direct co-culture between adipocytes, adipose tissue macrophages and adipose stem/progenitor cells (ASPCs) Understanding the range of interactions between adipocytes, tissue adipose macrophages and ASPCs is central to unlocking many of the cellular and molecular biological mechanisms of wound healing and adipose stem cell plasticity. Hypothesis: Direct cell to cell contact of adipocytes, adipose tissue macrophages and ASPCs generate (i) new preadipocytes in part through adipose tissue macrophages differentiation

Methods:
Human adipose tissue obtained from obese patients was treated with collagenase and adipocytes were isolated by centrifugation. Adipose tissue macrophages and ASPCs were isolated from the stromal vascular fraction using a Ficoll gradient. Adipocytes and the macrophage adipose tissue/ASPC fraction were cultured separately for 24 hours, co-cultured for a further 24 hours, and then separated and cultured alone for a further 48 hours. In some experiments the adipose tissue macrophage/ASPC fraction was incubated with fluorescent anti-human CD14 nanobeads. After separation of unbound or non-internalized nanobeads, the macrophage/ASPC fraction containing nanobeads was plated for 24 hours and co-cultured was performed as described above.

Results:
Approximately 90% of adipose tissue macrophage/ASPC fraction incorporated fluorescent anti-human CD14 nanobeads after internalization of these nanobeads. Co-culture between the CD14 nanobead-internalized macrophage/ASPCs fraction and the untreated adipocytes, majority of preadipocytes generated contained internalized CD14(+) nanobeads. The resulting CD14 nanobead labeled preadipocytes were also positive for S-100 and DLK confirming that most of the new preadipocytes were of CD14 precursor cell origin. These new preadipocytes were also CD34(+), CD105(+), and CD146(+). Preadipocyte proliferation required direct cell to cell contact, since proliferation was greatly reduced if co-culture was performed using a transwell system. Interestingly, we also observed after co-culture a population of tiny CD34 (+) ASPCs. These cells were also positive for S-100.

Conclusions:
Through co-culture of adipocytes, and adipose tissue macrophage and ASCPs fraction it is possible (i) to generate new preadipocytes derived from adipose tissue macrophages (ii) to generate a novel population of CD34 (+) ASPCs, that were also positive for markers commonly found in preadipocytes/adipocytes. These findings could have far reaching implications with adipocyte growth, wound healing and adipose stem cell plasticity modulated, at least in part, by this novel cellular differentiation pathway.

 


Abstract ID: 9

Differential Adipogenesis Related Gene Expression in Mesenchymal Stromal Cells derived from Bone Marrow, Cord Blood and Adipose Tissue.

Marianna Karagianni; Karen Bieback; Harald Klueter

Introduction:
Mesenchymal Stromal Cells (MSCs) from Bone Marrow (BM- MSCs) and Adipose Tissue (AT- MSCs) undergo in vitro differentiation into mesodermal derivatives such as adipose-, bone- and cartilage tissue. Cord Blood MSCs behave differently in response to adipogenic stimuli. They fail to develop the mature adipocyte phenotype with perinuclear lipid droplets and inversion of the nuclear-cytoplasmic relation. We tried to track specific protein markers and transcriptional factors during adipogenic induction of BM-, AT- and CB-derived MSCs to identify a potential inhibitory pathway in CB-MSCs.

Methods:
AT-, BM- and CB-MSCs were cultivated in MSC medium as undifferentiated condition. Differentiation towards the adipogenic lineage was induced by adipogenic stimuli (insulin, dexamethasone, isobutylmethylxanthine, indomethacin) after reaching postconfluency. The expression of lipid droplets associated proteins (LDAP) like adipophilin, and perilipin was followed by immunofluorescence (IF). Quantitative RT-PCR gave relative quantification comparisons on the time-dependent expression of transcriptional factors and genes such as PPARg, CEBPa, adiponectin and DLK/Pref-1. Knock-down of DLK1/Pref-1 was performed via si-RNA in CB-MSCs. qRT-PCR was performed subsequently for PPAR-g and adiponectin.

Results:
Following culture of BM-, AT- and CB-MSCs under adipogenic conditions, we found induction of the adipogenic phenotype and positive IF signals for perilipin, a specific LDAP for adipocytes, in BM- and AT-MSCs. This was associated with increased levels in mRNA expression of perilipin, c-EBP-a, PPAR-g and adiponectin. In contrast the phenotype of the CB-MSCs remained unaltered, the IF-detection of perilipin persisted negative. The m-RNA expression of perilipin, c-EBP-a, PPARg and adiponectin in CB-MSCs was decreased in relation to AT- and BM-MSCs. DLK/Pref-1, a transcriptional factor, associated with an undifferentiated state of preadipocytes, was downregulated in induced BM- and AT-MSCs. In treated CB-MSCs it remained unsuppressed, supporting the notion that DLK/Pref-1 behaves as a growth factor, maintaining the proliferative state of undifferentiated cells. Inhibition of DLK/Pref-1 via si-RNA in CB-MSCs resulted in an upregulation of PPAR-g and adiponectin mRNA. A mature adipogenic phenotype however was not achieved.

Conclusions:
CB-MSCs fail to undergo adipogenic differentiation in contrast to BM- and AT-MSCs. Our data indicate a correlation with the expression of DLK/Pref-1, which was shown to be unsuppressed in CB-MSCs in contrast to its downregulation in BM and AT-MSCs under adipogenic induction. mRNA interference with DLK/Pref-1-si-RNA in CB-MSCs corroborated our thesis, as it resulted an upregulation of PPAR-g and adiponectin -but still no adipogenic phenotype- in treated CB-MSCs. We suggest CB-MSC as a potent model for studying modulatory pathways of the adipogenic cascade, especially influenced by aging, with a focus on the inverse correlation between adipogenesis and osteogenesis, which are amongst others controlled by Pref-1 expression.

 


Abstract ID: 37

Toll-like receptor-mediated signaling in human adipose-derived stem cells: Implications on immunogenicity and immunosuppressive potential

Eleuterio Lombardo

Introduction:
Human adipose-derived stem cells (hASCs) are mesenchymal stem cells with reduced immunogenicity and the capability to modulate immune responses. These properties make hASCs of special interest as therapeutic agents in the settings of both chronic inflammatory and autoimmune diseases. Toll-like receptors (TLR) ligands, both exogenous and endogenous, have been linked with the perpetuation of inflammation in a number of chronic inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis, due to the permanent exposure of the immune system to TLR-specific stimuli. Therefore, hASCs employed in therapy are potentially exposed to TLR ligands which may result in the modulation of hASC activity and therapeutic potency.

Methods:
We investigated the role of TLR signalling in hASCs function in vitro. hASCs differentiation assays as well as lymphocyte proliferation assays were carried out. Expression of inflammatory cytokines upon stimulation was determined by cytometric bead arrays and ELISAS.

Results:
In this study we demonstrate that hASCs possess active TLR2, TLR3 and TLR4 as the activation with specific ligands resulted in induction of the NF- B-dependent gene manganese superoxide dismutase (MnSOD) and the release of IL-6 and IL-8. Whereas osteogenic differentiation was increased by TLR3 and TLR4 ligands, no effect on adipogenic differentiation or proliferation was observed. Moreover, we show that TLR activation does not impair the immunogenic and immunosuppressive properties of hASCs.

Conclusions:
hASCs are activated by TLR ligands. However, this activation does not affect the immunogenic and immunosuppressive abilities of hASCs. Therefore, these results may have important implications with respect to safety and efficacy of hASC-based cell therapies.

 

 


Abstract ID: 47

Expanded Adipose-derived Stem Cells (Cx401) for the Treatment of Complex Perianal Fistula. A Phase II Clinical Trial

Tommaso Rada; Rui L. Reis; Manuela E. Gomes

Introduction:
Management of complex fistulas is a challenge due to the limitations of current treatments. Expanded adipose derived mesenchymal Stem Cells (Cx401) is a novel cell therapy based on immunoregulation and cell proliferation which helps repair damaged tissue.

Methods:
A multicenter, randomized, controlled trial is being conducted to evaluate the efficacy and safety of Cx401 in 49 adult patients with complex perianal fistula from Cryptoglandular (35) or Crohn (14) diseases. Patients received fibrin glue or 20 million cells plus fibrin glue intralesionally. Fistula healing was evaluated at 8 wks. If not healed, a second dose of fibrin glue or 40 million cells plus fibrin glue was administered, with healing evaluated 8 wks later. Healing was defined as absence of drainage (spontaneous or by gentle compression) and complete re¬epithelization of the external openings.

Results:
The proportion of patients whose fistulas were healed was significantly higher with Cx401 than with fibrin glue [17(70.83%) vs 4(16.00%) respectively, RR=4.43(CI 1.74, 11.27);p¬value=0.0001]. Efficacy was observed in Crohn and non-Crohn. 45% of Cx401 patients received a cumulative dose of 60 million cells. At 6 months follow-up, 2 SAEs were observed in each group. Only one SAE was related to fibrin glue, no SAE was related to Cx401

Conclusions:
A dose of 20–60 million Expanded Adipose-derived Stem Cells (Cx401) in combination with fibrin glue is an effective and safe treatment for complex perianal fistula

 


Abstract ID: 42

Adipose Derived Adult Stem cells’ (ADAS) capacity to differentiate into Endothelial Cells expressing von Willebrand marker and forming capillary-like structures in a Skin Equivalent model.

Celine Auxenfans; Lauriane Thivillier; Charlotte Lequeux; Nicolas Bechetoille; Stephanie Maillet; Nathalie Tan; Beste Kinikoglu; Valérie André; Odile Damour

Introduction:
Adipose tissue is now recognized as a source of autologous stem cells with multilineage capacity, able to differentiate into various cell types, notably into endothelial cells [1, 2]. In cell therapy, its abundance and accessibility make it an alternative to bone marrow and peripheral blood, which are the only current sources of autologous endothelial progenitor cells (EPCs). Thus, this route broadens therapeutic perspectives on regenerative medicine, such as revascularization of ischemic tissues or repair of damaged vessel walls and vascularized tissue engineering (vascular prostheses and skin equivalents for the treatment of large burns). After optimizing culture conditions for the proliferation of the SVF cells and their differentiation into EPCs, the aim of this study was to demonstrate their functionality by the expression of von Willebrand Factor and their capacity to form tubular structures in a skin equivalent (SE) model. Indeed, our SE model was prepared from cocultured fibroblasts and keratinocytes in a chitosan cross-linked collagen-glycosaminoglycan (CGC) scaffold. Endothelialized skin equivalent was previously obtained by seeding both HUVECs and fibroblasts into the dermal part of this model. Under these conditions, capillary-like structures were formed in a coculture environment containing newly synthesized extracellular matrix by fibroblasts and keratinocytes [3]

Methods:
ADAS cells, differentiated along endothelial lineage by treatment with VEGF, were seeded together with fibroblasts into the CGC scaffold at the same density, and then cultured for 3 weeks in fibroblast medium supplemented with VEGF before seeding the surface with keratinocytes. After keratinocyte seeding, the model was cultured under submerged conditions for 1 week, then lifted to an air/liquid interface and cultured for 2 more weeks. The samples were then fixed in formalin and Tissue Tek for histology and immunohistology

Results:
The average percentages of CD 133+ and CD31+ cells, measured by flow cytometry on freshly isolated cell suspension, are 29% and 11%, respectively. At passage 1, before predifferentiation, they were 36% and 11%, respectively. Immunostaining shows that ADAS are able to form capillary-like structures in which cells express both von Willebrand marker and basal membrane components such as Laminin 5. No fluorescence was detected in the non-endothelialized control SE

Conclusions:
In the endothelialized skin equivalent (ESE), cell-cell and cell-matrix interactions, VEGF supplementation and long-term culture induce differentiation of preamplified ADAS cells into ECs that are able to aggregate into capillary like structures and express von Willebrand marker. These results suggest that adipose tissue may be considered as an accessible source of endothelial cells for cell therapy and ESE reconstruction. 1. Cao, Y., et al., Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun, 2005. 332(2): p. 370-9. 2. Planat-Benard, V., et al., Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation, 2004. 109(5): p. 656-63. 3. Black, A.F., et al., A novel approach for studying angiogenesis: a human skin equivalent with a capillary-like network. Cell Biol Toxicol, 1999. 15(2): p. 81-90.

 


Abstract ID: 116

Adipose Derived Stromal Cells Express Tissue Factor in a Passage-Dependent Manner: Implication for Intracoronary Cell Therapy and Use of Heparin with ASCs

Vineet Kumar; Malgorzata Maria Kamocka; Elliot D Rosen; Clayton A Smith; Brain Johnstone; Keith L March

Introduction:
Adipose stromal cells (ASCs) are readily available autologous cells with potential for treatment of cardiac disease. Several trials of ASC involve intravascular delivery, including intracoronary delivery, and accordingly we wished to determine whether ASC exhibit procoagulant (PC) properties

Methods:
Human ASC either before passage (P0) or after 1 or 2 passages in EGM2-MV, or else human endothelial cells (EC) at passages 2-3, as a control, were evaluated for PC activity in normal human plasma, using standard protocols for testing prothrombin time (PT) in the absence of exogenously added thromboplastin (purified tissue factor). Time to coagulation in standard 150 μl volumes was measured using change in viscosity by a Start 4 coagulometer (Diagnostica Stago). From 0 to 5x105 ASC, obtained from 3 different subjects and at the different passages, or EC were added to test vials. Assays were performed with cells alone; or in the presence of increasing concentrations of heparin, the factor X inhibitor fondaparinux, or with a neutralizing anti-human tissue factor antibody. Cell surface-exposed activated tissue factor (TF) was quantitated by comparing activity to that of known TF quantities. The expression of coagulation factors, such as TF, was also evaluated by Affymetrix gene chip analysis.

Results:
The time to coagulation increased with ASC concentrations and was inversely proportional to passage number. The PC activity of 5x105 P0 ASC was equivalent to 0.53% of the concentration of thromboplastin normally employed in the PT assay, and resulted in coagulation at 39.2 seconds. The same number of ASC at P1 and P2 exhibited, respectively, 0.29% and 0.05% of the standard thromboplastin activity. In the absence of thromboplastin or ASC, as well as in the presence of equivalent numbers of EC, there was no coagulation. The PC activity of ASC was inhibited by neutralizing tissue factor antibody and also in a dose-dependent fashion by heparin, with full inhibition at 1 U heparin/106 ASC. Indirect quantification of tissue factor expressed by ASC at P0, P1 and P2 indicated that 598, 328 and 56 picogram TF, respectively, was present on cell surface. The ASC at P2 consistently expressed tissue factor transcript at levels in the
upper quartile of all transcripts.

Conclusions:
ASC express TF in a passage dependent manner and may potentially increase thrombogenesis in vivo by activating the extrinsic coagulation pathway. The potent blockade of the activity by heparin suggests that admixture of heparin with ASC would be of potential utility for optimizing clinical safety, especially with of intracoronary delivery.

 


Mesenchymal stem cells for liver regeneration

Oscar K. Lee

We have demonstrated that extra-hepatic human mesenchymal stem cells (MSCs) differentiate into hepatocyte-like cells with functions sine qua non of normal hepatocytes, and that in utero transplantation (IUT) of MSCs can contribute to numerous organs including the liver. However, their therapeutic potential is questioned by the low frequencies of engraftment in IUT models as well as animal models of non-lethal liver injury. Here, we investigate the feasibility of rescuing lethal fulminant hepatic failure (FHF) with MSCs. The significance of a lethal model is that it reflects more closely the clinical demand for alternative or bridging therapies while waiting for available donor organs. FHF in non-obese diabetic severe combined immunodeficient mice was established by carbon tetrachloride (CCl4) administration, and MSCs were intrasplenically or intravenously transplanted. While placebo groups died from CCl4-injury, liver regeneration and long-term engraftment of donor-derived cells was demonstrated in MSC-recipients. Engraftment frequencies as well as comparison of MSCs with pre-differentiated hepatocytes-like cells indicate that rescue of FHF was not mediated by functional differentiation but, rather, by paracrine effects. We further found that undifferentiated MSCs express higher basal levels of anti-oxidative enzymes than pre-differentiated cells and, up-regulate anti-apoptotic genes under oxidative stress. It was also demonstrated that intravenous transplantations were more effective than intrasplenic transplantations. We propose that MSCs rescue FHF by scavenging and clearanceof reactive oxygen species, and promote endogenous liver regeneration through the release of trophic factors. Our pre-clinical results here demonstrate that mesenchymal stem cells can offer a novel therapy for lethal fulminant hepatic failure.

 


Abstract ID: 59

Microstructural Fat Grafting Improves Radiation Skin Damage in a Murine Model

Robert Allen, Jr.; Phuong Nguyen; Jeffrey Schachar; Vishal Thanik; Pierre Saadeh; Stephen Warren; Sydney Coleman; Alexes Hazen

Introduction:
Recent clinical reports have shown fat grafting to be a useful tool in the treatment of radiation fibrosis. The mechanism responsible for this clinical improvement, however, remains unknown. Since radiation fibrosis is known to be mediated by upregulation of the TGF-β/Smad3 pathway, we hypothesize that microstructural fat grafting improves radiation fibrosis by downregulating this fibrotic response and increasing the vascularization of these tissues.

Methods:
Lipoaspirate was harvested from healthy human donors. Dorsal skin of wild-type FVB mice was isolated and irradiated with 45Gy using a Varian 2300 Linear Accelerator. Four weeks following radiation, mice were either grafted with 1.5-cc of lipoaspirate or sham-grafted into the dorsal subcutaneous tissue. Hair growth, skin color, and degree of ulceration were analyzed photometrically. Fat graft survival was measured by volume displacement. Collagen production, fibrosis, and vascular density (% positive staining/hpf) of irradiated tissue was assessed via gomori trichrome, Smad3, and CD31 staining at 4 weeks post-grafting, respectively. Fat graft vascularization was analyzed by en bloc β-galactosidase staining of the fat graft and dorsal skin of tie2/lacZ transgenic mice at 4 weeks post-grafting.

Results:
Irradiated mice began to exhibit radiation damage at 13±2 days. Chronic ulceration and fibrotic skin thickening became stable 4 weeks post-irradiation. Fat grafts harvested at 4 weeks following injection had 82.7±5.3% survival. Hair regrowth, skin color/texture, and degree of ulceration were improved in fat-grafted mice compared to sham-treated controls when analyzed photometrically. Smad3 production was significantly decreased in treated animals (18.16±0.5% vs 29.34±0.7%, p<0.03). Collagen production subsequently had a 2.3-fold decrease in fat grafted mice compared to controls. Vascular density of irradiated skin was also increased in fat grafted mice (7.3±0.04% vs 5.2±0.09%, p<0.01). Examination of the fat-grafts on tie2/lacZ mice demonstrate neovascularization within the grafted fat that does not stain positive for β-galactosidase, signifying that these vessels do not have murine endothelial cells. There is also a clear point of inosculation between human and murine vasculature at the perimeter of the graft, with obvious perfusion of these vessels.

Conclusions:
Microstructural fat grafting improves radiation fibrosis. Four weeks following grafting, treatment animals have a decreased fibrotic response, with decreased expression of Smad3. This downregulation of the TGF-β/Smad3 pathway correlates with a decrease in collagen production. There is also a concomitant increase in the skin vascularity of these previously irradiated tissues. Furthermore, we have demonstrated for the first time that de novo vessels are forming from human elements within the fat grafts. There is directinosculation of vessels originating from the grafted tissue (human) to the host vasculature (murine). It is likely that this neovascularization establishes blood flow to the graft and improves perfusion to the surrounding tissues. It is probable that the combination of these mechanisms result in the improvement in radiation fibrosis following microstructural fat grafting.

 


Abstract ID: 62

Adipose derived stem cells accelerate primary nerve repair

Hiroshi Mizuno; Uysal A. Cagri; Hakan Orbay; Takahisa Okuda; Hiko Hyakusoku

Introduction:
Adipose derived stem cells (ASCs) were proven to have a positive effect on healing processes. In in vitro experimental models, ASCs were indicated to differentiate into different cell lineages including neurons. We have performed an in vivo experimental study to find out the effect of ASCs on primary nerve repair model.

Methods:
The ASCs were gathered from inguinal fat pads of fisher rats. After three passage in control medium (DMEM,10% FBS), the cells were labeled with DiI and Hoecht 33342 for tracing. 1x107cells were used in every application. The sciatic nerves of 6 rat were explored and sharp incision was performed before the branching into peroneal and tibial nerves. The nerve was coapted under operating microscope. The right side was covered with fibrin glue and ASCs and the left sciatic nerve was applied fibrin glue only. Walking gate analysis was performed on 3rd and 6th months. Electro neurography (ENG)was performed on the 6th month. Histological and immunohistochemical staining with anti S100 protein antibody and VEGF were done. SEM was performed.

Results:
Walking gate analysis at the 3rd month revealed that the sciatic function indexes (SFI) were -108.677±7.436, -83.061±8.183 in the control and experimental groups respectively. At the 6th month SFI was -83.246±7.352, -62.256±7.349 in the control and experimental groups respectively. The nerve conduction velocity (m/sec) was 34.88±5.15 in the control and 48.01±6.83 in the experimental group. The conduction percentage of the given voltage was 5.37%±1.89 in control and 9.33%±2.01 in the experimental group. There was statistical significance in all of the evaluation methods. (p<0.05) The immunohistochemical staining revealed the differentiation of the stem cells to endothelial and schwann cells. VEGF was increased in the experimental group. TGF beta1, TGF beta2, TGF beta 3 and FGF was significantly different between the groups.

Conclusions:
ASCs might help in the primary healing process of the nerve directly and indirectly. Direct effect is mainly the differentiation to endothelial cells and neural cells, mainly schwann cells, supported by the ENG and histologically. Indirectly, the differentiation to fibroblasts could increase the growth hormones and cytokines mainly and this indirect effect is the main controller of the healing process.

 


Abstract ID: 82

Assessing the Risk of Tumorigenesis Through the Interactions of Adipose Derived Stem Cells and Breast Cancer

William Austen Jr.; Mark Randolph; Raymond Wadlow; Mike McCormack; John Nguyen

Introduction:

Fat grafts supplemented with adipose derived stem cells (ADSC) have been shown to have improved graft blood supply as well as improved longevity and graft volume. The clinical feasibility of using adipose derived stem-cell supplemented fat for breast augmentation or reconstruction raises the issues of tumor initiation, promotion and progression. There is compelling evidence that stromal cells, including fibroblasts can influence epithelial transformation and are prominent modifiers of cancer progression. Little is known about the dynamics of interaction between tumors and therapeutic stem cells. The purpose of this study is to perform an examination of the cell-cell interaction and assess potential tumorigenesis between ADSC and breast cancer cells.

Methods:

To examine the behavior between immortalized tumors and mesenchymal stem cells we developed a uniform monolayer co-culture system. ADSC were isolated from over 20 patients and grown until confluent on the base of a 96 well plate. This layer of stem cells acts as a feeder layer and is seeded with various green fluorescence protein (GFP)-labeled breast cancer cell lines. Breast cancer cell lines included ER, PR, and HER2 positive and negative receptors. Analysis of GFP expression was performed daily using a fluorescence plate reader to assess important characteristics of GFP-expressing cancer cells such as viability, cell growth, and proliferation. ADSC and breast cancer cells showing increased GFP expression are FACs sorted, examined for expression of relevant surface markers, and further co-cultured with a semi-permeable membrane using minicell co-culture inserts.

Results:

We have enabled the stable transduction of the GFP gene into a large series of human breast tumor cell lines and the tumor cell lines were able to stably express GFP at high levels. The present study demonstrated that the presence of ADSC in co-culture with a benign mammary epithelial cell line remained unchanged in growth and proliferation in vitro as compared to fibroblasts controls and benign mammary epithelial cells alone. Additionally, co-culture of various human breast cancer cell lines remained unchanged in the presence of all but one ADSC of the patients examined. We have found in repeated experimentation that co-culture of a single patient’s ADSC (1/20) had significantly increased the growth and proliferation of one cancer cell line (1/5). There was an exponential increase in growth and proliferation as compared to fibroblast controls and breast cancer cell growth alone. Furthermore, 1 out 5 of our cancer cell lines exhibited an increase in growth and proliferation when co-cultured with our fibroblast control.

Conclusions:

The purpose of this study was to perform an examination of the cell-cell interaction and assess potential tumorigenesis between ADSC and breast cancer cells. We have developed a uniform monolayer co-culturing system that effectively allows us the ability to assess important characteristics of cancer cell growth and proliferation using GFP expression. Thus far, we have found a population of ADSC that when co-cultured with an ER positive breast cancer cell line, seems to stimulate an increase in growth and proliferation. This finding does not indicate that ADSC stimulate cancer but does suggest that there are patient phenotypes that may have a factor in tumorigenesis. The mechanism in which these ADSC enhances proliferation and growth in these breast cancer lines is unclear; however, examination of cells for expression of relevant surface markers, and further examination using this and other models is warranted.