Supernatants of Adipocytes From Obese Versus Normal Weight Women and Breast Cancer Cells: In Vitro Impact on Angiogenesis.

Breast cancer is correlated with a higher risk of metastasis in obese postmenopausal women. Adipokines, whose plasma concentrations are modulated in obese subjects and adipocytes surround mammary cells, suggesting that adipocyte secretome affect mammary tumorogenesis. We hypothesize that mature adipocyte secretions from obese women conditioned or not by breast neoplasic cells, increase changes on the angiogenesis stages. Supernatants of human mature adipocytes, differentiated from stem cells of either adipose tissue of normal weight (MA20) or obese (MA30) women or obtained from co-cultures between MA20 and MA30 and breast cancer cell line MCF-7, were collected. The impact of these supernatants was investigated on proliferation, migration, and tube formation by endothelial cells (HUVEC). MA20 and MA30 showed a preservation of their "metabolic memory" (increase of Leptin, ObR, VEGF, CYP19A1, and a decrease of Adiponectin expression in MA30 compared to MA20). Supernatants from obese-adipocytes increased HUVEC proliferation, migration, and sprouting like with supernatants obtained from co-cultures of MA/MCF-7 regardless the women's BMI. Additional analyses such as the use of neutralizing antibodies, analysis of supernatants (Milliplex®) and variations in gene expression (qRT-PCR), strongly suggest an implication of IL-6, or a synergistic action among adipokines, probably associated with that of VEGF or IL-6. As a conclusion, supernatants from co-cultures of MA30 and MCF-7 cells increase proliferation, migration, and sprouting of HUVEC cells. These results provide insights into the interaction between adipocytes and epithelial cancer cells, particularly in case of obesity. The identification of synergistic action of adipokines would therefore be a great interest in developing preventive strategies. J. Cell. Physiol. 232: 1808-1816, 2017. © 2016 Wiley Periodicals, Inc.

Intradermal injection of human adipose-derived stem cells accelerates skin wound healing in nude mice.

BACKGROUND: The use of stem cells from adipose tissue or adipose-derived stem cells (ASCs) in regenerative medicine could be an interesting alternative to bone marrow stem cells because they are easily accessible and available in large quantities. The aim of this study was to evaluate the potential effect of ASCs on the healing of 12 mm diameter-excisional wounds (around 110 mm(2)) in nude mice. METHODS: Thirty nude mice underwent surgery to create one 12-mm excisional wound per mouse (spontaneous healing, n = 6; Cytocare® 532, n = 12; ASCs, n = 12). The Galiano wound model was chosen to avoid shrinkage and thus slow the spontaneous healing (SH) of mouse skin, making it closer to the physiology of human skin healing. Transparent dressings were used to enable daily healing time measurements to be taken. Immunohistochemistry, histological and blood perfusion analysis were carried out on the healed skin. RESULTS: The in vivo results showed the effectiveness of using ASCs on reducing the time needed for complete healing to 21.2 days for SH, 17.4 days for vehicle alone (Cytocare® 532) and 14.6 days with the addition of ASCs (p < 0.001). Moreover, cutaneous perfusion of the healed wound was significantly improved in ASC-treated mice compared to SH group, as shown by laser Doppler flowmetry and the quantitation of blood vessels using immunohistochemistry of αsmooth muscle actin. CONCLUSIONS: The tolerance and efficacy of cryopreserved ASCs to accelerate the complete closure of the wound by increasing the maturation of the skin and its blood perfusion, shows their therapeutic benefit in the wound healing context.

In vitro and in vivo biocompatibility, bioavailability and tolerance of an injectable vehicle for adipose-derived stem/stromal cells for plastic surgery indications.

Soft tissue reconstruction is a challenge in plastic surgery, when replacing lost materials and correcting contour defects. Many permanent and temporary fillers have been used to restore the volume of these lesions, but often with poor results and even complications. Adipose-derived stem/stromal cells (ASCs) and adipose tissue engineering have been suggested as valuable alternatives. In order to inject these cultured cells, it was essential to find a suitable vehicle. The purpose of this study was to evaluate Cytocare(®), an injectable medical device, composed of hyaluronic acid plus amino acids, vitamins and mineral salts. First, ASC viability and bioavailability in the 3 different available Cytocare(®) formulations using the MTT test were assessed; then an animal experiment, testing the tolerance after intradermal injections of both Cytocare(®) alone and with ASCs was carried out. Our in vitro results demonstrate a high biocompatibility of Cytocare(®) resulting in a better viability of ASCs when cultured in Cytocare(®) compared to culture medium (p < 0.05, Mann and Whitney). Cytocare(®) also permits their bioavailability and proliferation, making it a potential transfer vehicle that can retain the cells before their integration around the recipient site. Finally, our animal experiment shows that the ASC + Cytocare(®) combination is well tolerated. In conclusion, Cytocare(®) can be used as a biocompatible scaffold for cultured ASCs in therapeutic treatments, ensuring ASC bioavailability, as well as evidence of excellent tolerance in nude mice.

Reciprocal interactions between breast tumor and its adipose microenvironment based on a 3D adipose equivalent model.

Breast cancer has become the most common cancer among women in industrialized countries. Obesity is well established as a risk factor, in particular owing to the attendant secretion of the entities called adipokines; there is growing evidence for a role of cells and factors present in the mammary tumor microenvironment such as fibroblasts, preadipocytes, adipocytes and their secretions. To study how the microenvironment influences breast cancer growth, we developed a novel tridimensional adipose model epithelialized with normal human keratinocytes or with breast cancer cell lines. These mimicked a breast tumor in contact with an adipose microenvironment and allowed monitoring of the interactions between the cells. Leptin and adiponectin, two major adipokines, and their respective receptors, ObRt and AdipoR1, were expressed in the model, but not the second adiponectin receptor, AdipoR2. The differentiation of preadipocytes into adipocytes was greater when they were in contact with the breast cancer cell lines. The contact of breast cancer cell lines with the microenvironment completely modified their transcriptional programs by increasing the expression of genes involved in cell proliferation (cyclinD1, MAPK), angiogenesis (MMP9, VEGF) and hormonal pathways (ESR1, IL6). This tridimensional adipose model provides new insights into the interactions between breast cancer cells and their adipose microenvironment, and provides a tool to develop new drugs for the treatment of both cancer and obesity.

Transdermal delivery of adipocyte-derived stem cells using a fractional ablative laser.

BACKGROUND: Chronic wound healing problems can pose a significant clinical challenge. Transdermal delivery of adipose-derived stem cells (ADSC) may be a possible solution to healing these recalcitrant, debilitating wounds. Pretreatment of the skin with a fractionated laser has already been shown to assist transdermal drug delivery both in vitro and in vivo and may be an ideal approach to facilitating delivery of ADSC to the target tissue. OBJECTIVES: The authors investigate in a porcine model whether ADSC can be delivered transdermally following pretreatment with a fractional laser. METHODS: After ethics approval was obtained, the abdomens of 2 adult female domestic pigs were pretreated with an erbium:YAG fractionated ablative laser. Following laser treatment, 20 × 10(6) bromodeoxyuridine (BrdU)-labeled ADSC were applied topically to the first animal for 4 hours. The same number of BrdU-labeled ADSC was applied to the second animal for 48 hours. The animals were euthanized at the end of their respective treatment periods, and the BrdU-labeled ADSC were counted after tissue harvest. RESULTS: At 4 hours, an average of 2.40 × 10(6) cells, or 12.0% of the total cells applied, were found in the tissue. At 48 hours, an average of 1.1 × 10(6) cells, or 5.5% of the total cells applied, were seen. CONCLUSIONS: This pilot study demonstrates that ADSC can be delivered transdermally through skin that has been pretreated with a laser. Potential future applications of this approach might include wound-healing or aesthetic indications. Further studies need to be conducted to determine the optimal number of ADSC to use in this approach, the best methods of application, and the effect of transdermally delivered ADSC on wound healing.

Subcutaneous fat tissue engineering using autologous adipose-derived stem cells seeded onto a collagen scaffold.

BACKGROUND: This pilot study examined the efficacy of 5-bromo-2-deoxyuridine-labeled autologous adipose-derived stem cells seeded onto collagen scaffolds to augment and/or regenerate the fat-enriched hypodermal tissue in an acute porcine wound model. METHODS: Porcine autologous adipose-derived stem cells were isolated and cultured. At passage 2, the cells were labeled with 5-bromo-2-deoxyuridine, seeded onto a three-dimensional collagen scaffold, and cultured for 10 days. Scaffolds were implanted subcutaneously in adult pigs with two adipose-derived stem cell scaffolds and two control scaffolds. Animals were euthanized at 2, 4, 8, and 12 weeks; all scaffold conditions were explanted for histology and immunohistochemistry analyses. RESULTS: For all time points, adipose-derived stem cell scaffolds had increased connective tissue matrix within the subcutaneous tissue compared with scaffold alone and untreated porcine skin (p < 0.01). The neosynthesized connective tissue was vascularized and composed of small cells within an abundant extracellular matrix organized in layers. 5-Bromo-2-deoxyuridine cells were detectable only up to 4 weeks and mature adipocytes were absent. Levels of collagen types I, III, and VI differed among the experimental groups, with increased extracellular matrix associated with the presence of adipose-derived stem cells. CONCLUSIONS: The authors' data clearly show the efficacy of adipose-derived stem cells for soft-tissue repair and skin aging because it induces a significant increase of the dermis thickness. Moreover, the authors' results demonstrate the interest of their acute wound model and allowed them to show the skin thickness variation over time of the experiment, which is one of the challenges with which clinicians struggle in fat grafting.

Influence of age and body mass index on the yield and proliferation capacity of adipose-derived stem cells.

BACKGROUND: Adipose tissue is commonly used for volume restoration. It is also a source of adipose-derived stem cells (ASCs), easy to obtain in large quantities by liposuction or resection techniques. The aim of this study was to determine the influence of body mass index (BMI) and age on the number (yield) and proliferation capacity of ASCs. METHODS: A prospective study was conducted in 42 women. They were divided into two groups: age ≤ 40 or >40 and BMI ≤ 25 or >25. Fat tissue was harvested via manual lipoaspiration always from the abdominal region. After centrifugation in the OR, the harvested fat (100 cc) was sent to the laboratory for isolation and cultivation of ASCs. The yield of viable ASCs was evaluated by the trypan blue exclusion test. Viable ASCs were cultured and their proliferation capacity was evaluated by the growth kinetics assay. Results were statistically analyzed. RESULTS: The average cell yield was 0.380 × 10(6)/ml. Cell yield and proliferation capacity did not show statistically significant correlation to the age and BMI of patients, with regression lines showing null correlation. There was no significant difference between the cell yield and proliferation capacity between the different groups. CONCLUSION: The results from this study suggest that there is no statistically significant correlation between ASC yield and proliferation capacity and age and BMI.

Stem cells, mature adipocytes, and extracellular scaffold: what does each contribute to fat graft survival?

BACKGROUND: Soft tissue engineering offers new perspectives for improving fat graft survival, for which the appropriate association of cells and scaffold seems essential. This study aimed to analyze the survival of free-cell grafts compared with adipose-derived stem cells (ASCs) seeded on collagen scaffolds. METHODS: Adipose tissue from a single volunteer was used for the following preparations: purified adipose tissue, isolated mature adipocytes (free-cell graft), cultured ASCs without scaffold (free-cell graft), collagen scaffold only, cultured ASCs in collagen scaffold without and with bioactive factors, and freshly-isolated ASCs in collagen scaffold. These were grafted on 18 nude mice for 2 months, after which specimens were evaluated grossly and histologically using hematoxylin-phloxine-safran (HPS), Oil-Red-O, and antivimentin labeling. Specimens and animals were weighed before implantation and after explantation, and weight values were statistically analyzed. RESULTS: Free-cell grafts (mature adipocytes and free ASCs) showed complete resorption in 50 and 60% of the animals (remaining weight fraction was 22.5 and 5.3%, respectively). The survival of purified adipose tissue was 81.8% (statistically greater compared with free-cell grafts; p < 0.05). In the ASCs-scaffold association, the remaining weight fractions (87.3-70.4%) were statistically greater than in free-cell grafts (5.3-22.5%; p < 0.05), but the difference between ASC-scaffolds and fat grafts was not statistically significant. These results were confirmed by clinical and histologic observations. CONCLUSION: Three-dimensional collagen scaffolds seem to improve survival of ASCs compared with free-cell grafts (adipocytes and free ASCs).

Adipose derived stem cells: efficiency, toxicity, stability of BrdU labeling and effects on self-renewal and adipose differentiation.

5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular stability, and potential effects on ASC differentiation. Porcine and human ASC (pASC and hASC, respectively) were labeled with BrdU at 5 or 10 µM for 2, 6, 24, and 48 h. BrdU toxicity and stability over time in monolayer cultures, in 3-D collagen scaffolds implanted to a porcine model and after thawing from long-term storage were evaluated by MTT assays and immunohistochemistry. ASC differentiation was evaluated by Oil Red O staining. BrdU was not cytotoxic at all tested concentrations and incubation times. BrdU color intensity within each cell and the number of ASC labeled with BrdU decreased as a function of both incubation time and BrdU concentrations. Labeling intensities decreased over time and were undetectable after 6 passages for pASC and 4 passages for hASC. In 3-D scaffolds, BrdU-labeled ASC were identifiable after 90 days of in vitro cultures and for 30 days in a porcine model. BrdU did not prevent preadipocyte differentiation and BrdU labeling was still detectable after subsequent thawing after long-term storage of ASC. BrdU is an excellent candidate reagent to label and track ASC that will allow distinction between BrdU-labeled donor cells and host cells. The data provides a foundation for conducting future tissue engineering projects using BrdU-labeled ASC.

Improvement of skin quality after fat grafting: clinical observation and an animal study.

BACKGROUND: Restoration of volume and contour defects is a challenge in plastic surgery. Autologous fat tissue transfer is gaining increasing popularity in this field. The aim of this study was to investigate the histologic modifications of the skin after fat tissue grafting on an animal model. METHODS: Thirty nude mice, divided into three groups, were used in the experiment. All 30 mice received human fat tissue on one side. On the opposite side, 10 mice received nothing (negative control group), 10 mice received cell proliferation medium, and the remaining 10 mice received only subcutaneous tunneling. Eight weeks later, biopsies of the skin and subcutaneous tissue were performed and specimens were analyzed by hematoxylin-phloxin-saffron staining. Dermis thickness was measured. To differentiate human from murine collagen fibers, human and murine collagen type I antibodies were used. The other types of collagen were investigated by immunohistochemistry (immunostaining) using collagen type III, V, and VI antibodies. RESULTS: Fat tissue was found in all animals. Macroscopically, fat tissue presented normal aspects, with abundant peripheral neovascularization. Histologic examination showed abundant extracellular matrix around the injected human fat tissue. This was attributable to increased type I collagen fibers of murine origin as a result of the murine fibroblast stimulation by the grafted human fat tissue. Dermal thickness after fat grafting was significantly greater. This was not attributable to inflammatory reactions, because no modification was detected in our control groups. CONCLUSIONS: This study shows that fat tissue grafting stimulates a neosynthesis of collagen fibers at the recipient site and makes the dermis thicker. However, the long-term effects remain undetermined and need further investigation.

Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering.

Since the culture of keratinocytes on feeder layers, research to produce skin equivalents has been motivated by the challenge of treating large burns and chronic wounds and by European regulations which both require proof of the innocuousness and the effectiveness of cosmetic products, and which forbid animal testing. The dynamism in fundamental research, dermocosmetology and the pharmaceutical industry has led to the evolution and complexification of reconstructed skin. The Collagen-GAG-Chitosan sponge, as well as the self-assembly model, allow dermal reconstruction in which the neosynthesized extracellular matrix contains all of the desired macromolecules. It is deposited forming an ultrastructurally organised architecture. The quality of the dermis obtained allows the development and regeneration of a pluristratified and differentiated epidermis firmly anchored by an organised dermal-epidermal junction. Evolution of reconstructed skin into models which are more and more similar to the physiological skin results in higher graft take rates in the treatment of burns and chronic wounds, and brings to research, to dermocosmetology and to the pharmaceutical industry, a wide range of products such as pigmented, endothelialized, immunocompetent, and now adipose reconstructed skins. The present review will mainly concentrate on the latest developments in skin engineering and will mostly concern the studies carried out by our groups.