Characterization of human adipose-derived mesenchymal stem cells in an in vitro inflammation model

J Platas1, V Mirabet2, MJ Alcaraz1, I Guillén1,3. 1University of Valencia, Department of Pharmacology and IDM 46100, Spain, 2Generalitat Valenciana, Transfusion Center 46014, Spain, 3Cardenal Herrera University CEU, Department of Chemistry, Biochemistry and Molecular Biology 46113, Spain

Introduction:

Adipose tissue is associated with a variety of inflammatory diseases. Human adipose tissue is an important source of multipotent adult stem cells (MSC), called mesenchymal cells from adipose tissue (AMSC), which are located in the perivascular area closely associated to other cell types. MSC are involved in regenerating damaged tissue and in immunomodulatory phenomena. However, the study of AMSC is controversial because of difficulties to establish their phenotype. The purpose of this study was to characterize the behaviour of AMSC in an in vitro model of inflammatory response in the presence of pro-inflammatory cytokines.

Material y Methods:

Adipose tissue from 5 healthy individuals undergoing lipectomy was obtained with the patient’s consent. The fat was crushed and treated with collagenase (1.5 mg/ml). The cell suspension was centrifuged and the cellular pellet was filtered and washed with culture medium containing 1% of antibiotics. Cells were cultured in DMEM/F12 supplemented with 15% human serum and 1% of antibiotics at 37°C and 5% CO2 until semi-confluence with renewal of the medium every 72h. Initial and subcultures cells were characterized by flow cytometry using antibodies against mesenchymal markers. For the selection of antibody markers we used the criteria established by the International Society for Cellular Therapy. AMSC cells are characterized by >95% of CD105+, CD90+ and CD13+ cells, and ≤2% of CD45- and CD34- cells, which are characteristic of hematopoietic cells. AMSC cells were stimulated with interleukin (IL)-1β (10 ng/ml) in the presence or absence of high mobility group box-1 (HMGB-1, 25 ng/ml) for 24h. We evaluated the production of prostaglandin E2 (PGE2), nitrite and matrix metalloproteinase (MMP) activity by fluorometric methods, and IL-6 and IL-10 by ELISA. Results were analyzed by one-way ANOVA followed by Dunnett’s t-test, *p<0.05; **p<0.01 respect to nonstimulated cells; #p<0.05 respect to IL-1β.

Results:

Our results indicate that cell population was ≥ 95% CD90+, CD105+ and CD13+ and <2% CD45- along the entire culture period, indicating a phenotype of MSC . However, the results also showed that at initial passage 0 we obtained more than 21% of cells positive for the negative marker CD34 which decreased until passage 3 where we found only 1% CD34+ cells according to international criteria of MSC. IL-1β significantly induced the production of inflammatory mediators PGE2, IL-6, IL-10 and MMP activity respect to basal conditions (72±3** vs 6±1, 1012±77** vs 335±166,123±22* vs 78±19 pg/mg protein, respectively, and 39982±164** vs 19201±148 FU/mg protein) in AMSC cells. However, IL-1β inhibited NO production in these cells (80798±144* vs 78119±148 FU/mg of protein). In addition, HMGB-1 significantly potentiated the effects of IL-1β on the production of PGE2 (136±11 # vs 72±3 pg/mg of protein).

Conclusions:

The phenotype of cells characterized by flow cytometry indicates a profile of expression of cell surface markers characteristic of MSC. IL-1β increases the production of inflammatory mediators and decreases the production of NO, which can be potentiated by HMGB-1. Our data indicate that AMSC, in addition to their role in tissue regeneration, may contribute to modulate the inflammatory process.