Adipose MSCs response to breast cancer cell-derived factors in conditioned media and extracts.

Adipose-derived mesenchymal stem cells (ASCs) are known to have immunomodulatory properties through soluble factors or by direct cell-to-cell contact. This study aimed to assess the expression of HLA-G and IDO activity in breast cancer and normal ASCs and to see whether ASC is capable of modulating both tumor cells and immune system cells in vitro. ASCs were enzymatically isolated from 15 breast cancer patients and 10 normal individuals. Then they were cultured, and the impact of their conditioned media on the movement of the MDA-MB-231 breast cancer cell line was studied in wound healing scratch assay. Next, PBLs from the peripheral blood of normal individuals were separated and co-cultured with breast cancer and normal ASCs. PBLs proliferation and apoptosis were assessed using CFSE labeling dye and annexin V/7AAD staining, respectively. IDO activity and HLA-G protein expression in ASCs were examined using kynurenine assay and Western blotting, respectively. Tumor-derived ASCs, especially those from higher stages of breast cancer, have stronger effects on the proliferation and movement of MDA-MB-231 cells than normal ASCs (P-value < 0.05). Apoptosis in PBLs increased in the presence of ASCs compared to PBLs cultured alone (P-value < 0.05). In contrast, necrosis of PBLs decreased in the presence of ASCs compared to apoptosis in these cells (P-value < 0.001). Collectively, ASCs may have strategic effects on both tumor cells and cells of the immune system in the tumor microenvironment, resulting in tumor development, growth, and metastasis.

Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

Within the tumour stroma, a heterogeneous population of cell types reciprocally regulates cell proliferation, which considerably affects the progression of the disease. In this study, using tumour conditioned medium (TCM) derived from breast tumour cell lines - MCF7 and MDA MB 231, we have demonstrated the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into tumour-associated fibroblasts (TAFs). Since the Wnt signalling pathway is a key signalling pathway driving breast tumour growth, the effect of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) was also examined. The response of ADSCs to TCM and sFRP4 treatments was determined by using cell viability assay to determine the changes in ADSC viability, immunofluorescence for mesenchymal markers, glucose uptake assay, and glycolysis stress test using the Seahorse Extracellular Flux analyser to determine the glycolytic activity of ADSCs. ADSCs have been shown to acquire a hyper-proliferative state, significantly increasing their number upon short-term and long-term exposure to TCM. Changes have also been observed in the expression of key mesenchymal markers as well as in the metabolic state of ADSCs. SFRP4 significantly inhibited the differentiation of ADSCs into TAFs by reducing cell growth as well as mesenchymal marker expression (cell line-dependent). However, sFRP4 did not induce further significant changes to the altered metabolic phenotype of ADSCs following TCM exposure. Altogether, this study suggests that the breast tumour milieu may transform ADSCs into a tumour-supportive phenotype, which can be altered by Wnt antagonism, but is independent of metabolic changes.

Mo1706 Local Application of Adipose-Derived Mesenchymal Stem Cells Can Lead to Intestinal Fistula Healing - Experimental Study

Effects of nebulized adipose-derived mesenchymal stem cells on acute lung injury following smoke inhalation in sheep

Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31-/CD45-), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance: Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.

Oxidative stress leads to the degeneration of retinal pigment epithelial (RPE) and photoreceptor cells. We evaluated the potential of adipose-derived mesenchymal stem cells (ASCs) as a therapeutic tool by studying the migration capacity of ASCs in vitro and their protective effect against RPE cell death under oxidative stress in vitro and in vivo. ASCs exhibited enhanced migration when exposed to conditioned medium of oxidative stressed RPE cells obtained by hydrogen peroxide. Migration-related axis SDF-1/CXCR4 was studied, and upregulation of SDF-1 in stressed RPE and of CXCR4 in ASCs was detected. Moreover, ASCs' conditioned medium prevented H2O2-induced cell death of RPE cells. Early passage ASCs had high expression level of HGF, low VEGF levels, and unmodulated IL-1β levels, compared to late passage ASCs. Thus, early passage ASCs show the potential to migrate towards damaged RPE cells and protect them in a paracrine manner from cell death induced by oxidative stress. In vivo, mice received systemic injection of NaIO3, and 72 h later, ASCs were transplanted in the subretinal space. Seven days after ASC transplantation, the eyes were enucleated fixed and frozen for immunohistochemical analysis. Under such conditions, ASC-treated mice showed preservation of nuclear layers in the outer nuclear layer and stronger staining of RPE and photoreceptor layer, compared to PBS-treated mice. Taken together, our results indicate that ASCs are able to home in on damaged RPE cells and protect against damage to the RPE and PR layers caused by oxidative stress. These data imply the potential that ASCs have in regenerating RPE under oxidative stress, providing the basis for a therapeutic approach to retinal degeneration diseases related to oxidative stress that could help save the eyesight of millions of people worldwide.

Mo1707 Serial Treatment With Anti-TNF Ab Reduces Tumor Burden in a Murine Model of Chronic Colitis, Compared to Single Dose Anti-TNF

Background: The effects of radiation on the cellular compartments of the tumor microenvironment (TME) might be essential in radiotherapy outcomes.Objective: We aimed to assess the effects of the different doses of gamma irradiation on viability, ABCA1 and MMP-9 expression in adipose-derived mesenchymal stem cells (ASCs) as a critical part of TME.Material and Methods: In this experimental study, ASCs were extracted from five healthy donors and irradiated with different doses of 5, 10 and 30 Gy of gamma. Then, RNA was extracted fromirradiated ASCs and cDNA was synthesized. The viability of ASCs was determined at 24, 48, 72 and 168 h after irradiation using trypan blue staining.The expression of ABCA1 was checked by quantitative real-time (qRT)-PCR technique and the expression of MMP-9 protein was evaluated by western-blot. Results: Based on our findings, 10 Gy and 30 Gy but not 5 Gy of gamma irradiation significantly decreased the viability of ASCs after 24, 48, 72 and 168 h compared tothe non-irradiated cells (P< 0.05). However, a dose of 5 Gy increased ABCA1 in ASCs significantly compared to 10 Gy and 30 Gy (P=0.01 and P=0.02, respectively).In addition, the analysis of western blot data showed that 5 Gy of gamma irradiation significantly increased the expression of MMP-9 in ASCs (P=0.019).Conclusion: It is concluded that various doses of gamma radiation elicit differential ASCs responses that may lead to different tumor cell reactions to the radiotherapy through bystander effects.

The use of fat grafting has expanded to include cell and tissue regeneration, necessitating investigations to ensure the viability of stromal and adipose-derived mesenchymal stem cells (ASCs) within the transferred fat parcels. This study explored the impact of harvesting technique and centrifugation on the viability of stromal cells and ASCs in lipoaspirate. Fat was harvested from patients undergoing fat grafting using 2 types of liposuction cannula: (A) a 3-mm blunt tip cannula with 3 smooth holes and (B) a 2.4-mm, sharp point port, multihole blunt tip cannula. Fat from cannula B underwent different processing methods: no centrifugation, 300g, 600g, and 900g centrifugation. Stromal cells were isolated, quantified, and evaluated for viability. ASCs were cultured from these samples to confirm survival. Lipoaspirates from 21 patients were analyzed. The mean stromal cell counts were 0.937 × 109 ± 0.346 × 109/mL for cannula A and 0.734 × 109 ± 0.266 × 109/mL for cannula B (P = 0.684), with viabilities of 98.79% and 98.22% (P = 0.631), respectively. ASCs isolated and after 2-passage culture were also higher for cannula A. Stromal cell quantification and viability were lowest in the noncentrifuged group (P < 0.05) and highest in the 600g centrifugation group. Fat harvesting using cannulas A and B showed no significant difference in stromal cell yield or viability. Handheld syringe liposuction preserved stromal vascular fraction cell and ASC viability. Centrifugation at different speeds did not significantly affect stromal cell viability.

The inhibitory influence of adipose tissue-derived mesenchymal stem cell environment and Wnt antagonism on breast tumour cell lines.

BackgroundAging skin is characterized by a disturbed structure and lack of blood supply, which makes it difficult to heal once injured. ADSCs secrete large amounts of cytokines, which promote wound healing and vascular regeneration through paracrine secretion, and the number of cytokines can be elevated by hypoxic pretreating. However, the components of ADSCs are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel synthesized from gelatin and has recently emerged as a potentially attractive material for tissue engineering applications. GelMA loaded with concentrated hypoxic pretreated ADSCs conditioned medium could provide a new method of treating wounds in aged skin.MethodsPrimary ADSCs were isolated from human adipose tissue and characterized by flow cytometry and differentiation test. ADSCs in passages 4-6 were pretreated in the hypoxic and normoxic environments to collect conditioned medium, the conditioned medium was then concentrated to prepare concentrated ADSCs conditioned medium(cADSC-CM)(the one collected from ADSCs under hypoxia was called hypo-CM ,and the one from normoxia was called nor-CM). The concentration of cytokines was detected. After treated with cADSC-CM, the abilities of proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs) were assayed, and Akt/mTOR and MAPK signal pathway was detected using western blotting. GelMA+hypo-CM hydrogel was prepared, and a comprehensive evaluation of morphology, protein release efficiency, degradation rate, mechanical properties, and rheology properties were performed. Full-thickness skin wounds were created on the backs of 20-month-old mice. After surgery, GelMA, GelMA+F12, GelMA+hypo-CM, and GelMA+nor-CM were applied to the wound surface respectively. H&E, Masson, and immunohistochemistry staining were performed, and a laser Doppler perfusion imager was used to evaluate the blood perfusion. The student’s t-test was used for analysis between two groups and a one-way analysis of variance (ANOVA) was used for analysis among multi groups.ResultsOur results revealed that 1) wounds in aged skin healed more slowly than that in young skin and exhibited poorer perfusion; 2) hypoxic pretreated ADSCs secreted more cytokines including VEGF by activating HIF1α; 3) hypo-CM promoted proliferation and migration of HUVECs through VEGF/Akt/mTOR and MAPK signal pathway; 4) GelMA-hypoCM accelerated wound healing and angiogenesis in aged skin in vivo.ConclusionGelMA loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells conditioned medium could accelerate wound healing in aged skin by promoting angiogenesis.

Decision letter: Osteosarcoma-enriched transcripts paradoxically generate osteosarcoma-suppressing extracellular proteins

Editor's evaluation: Osteosarcoma-enriched transcripts paradoxically generate osteosarcoma-suppressing extracellular proteins

Keloid, scar caused by atypical wound repair, represents a significant difficulty for specialists in plastic surgery and dermatology. Adipose-derived mesenchymal stem cells (ADSCs) can regulate fibrotic phenotypes of keloid fibroblasts (KFs) in a paracrine fashion, but whether small extracellular vesicles (SEVs) are the key functional carrier in ADSC paracrine regulation of KFs remains unknown. This study aims to explore whether the regulatory effects of conditioned medium (CM) obtained from ADSCs on KFs can be impaired by decreased SEV content in the ADSC-CM. Clinical specimens were utilized to extract keloid fibroblasts (KFs), normal fibroblasts (NFs), and adipose-derived stem cells (ADSCs). Fibroblasts were cultured with CM obtained from ADSCs untreated or treated with the sphingomyelinase inhibitor GW4869. The features of SEVs derived from ADSC-CM were characterized, and fibroblast proliferation, migration, apoptosis, and expression of ECM proteins were analyzed. The sphingomyelinase inhibitor GW4869 successfully reduced the SEV content in ADSC-CM, and both control ADSC-CM and ADSC-CM with reduced SEV content significantly inhibited KF proliferation, migration, and α-SMA synthesis but not KF apoptosis, whereas only NF proliferation was inhibited by ADSC-CM. The reduced SEV content only affected the inhibition of KF proliferation induced by ADSC-CM. ADSC-CM inhibits various fibrotic phenotypes of KFs, but decreasing the SEV content in ADSC-CM did not significantly alter the antifibrotic effects of ADSC-CM. Thus, SEVs may not be the key mediator of ADSCs paracrine regulation of KFs. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors . www.springer.com/00266 .