An Evaluation of the Stemness, Paracrine, and Tumorigenic Characteristics of Highly Expanded, Minimally Passaged Adipose-Derived Stem Cells.

The use of adipose-derived stem cells (ADSC) in regenerative medicine is rising due to their plasticity, capacity of differentiation and paracrine and trophic effects. Despite the large number of cells obtained from adipose tissue, it is usually not enough for therapeutic purposes for many diseases or cosmetic procedures. Thus, there is the need for culturing and expanding cells in-vitro for several weeks remain. Our aim is to investigate if long- term proliferation with minimal passaging will affect the stemness, paracrine secretions and carcinogenesis markers of ADSC. The immunophenotypic properties and aldehyde dehydrogenase (ALDH) activity of the initial stromal vascular fraction (SVF) and serially passaged ADSC were observed by flow cytometry. In parallel, the telomerase activity and the relative expression of oncogenes and tumor suppressor genes were assessed by q-PCR. We also assessed the cytokine secretion profile of passaged ADSC by an ELISA. The expanded ADSC retain their morphological and phenotypical characteristics. These cells maintained in culture for up to 12 weeks until P4, possessed stable telomerase and ALDH activity, without having a TP53 mutation. Furthermore, the relative expression levels of TP53, RB, and MDM2 were not affected while the relative expression of c-Myc decreased significantly. Finally, the levels of the secretions of PGE2, STC1, and TIMP2 were not affected but the levels of IL-6, VEGF, and TIMP 1 significantly decreased at P2. Our results suggest that the expansion of passaged ADSC does not affect the differentiation capacity of stem cells and does not confer a cancerous state or capacity in vitro to the cells

List of primer sequences for real-time PCR.

<p>List of primer sequences for real-time PCR.</p

Immunophenotype and viability of the stromal vascular fraction (SVF).

<p>The cell viability was confirmed by flow cytometry analysis. (A) The viability of the stromal vascular fraction cells. (B) The flow cytometry histograms of the SVF for a representative donor are displayed. The percentage of cells that stained positive is indicated in the upper right corner of each panel. The green line indicates the positively stained cells, whereas the purple line indicates the isotype-matched monoclonal antibody control.</p

The paracrine activity of ADSCs during passaging.

<p>The levels of immunomodulatory proteins (A) PGE₂ and (B) IL-6; (C) the anti-apoptotic protein STC1; and the trophic proteins (D) VEGF, (E) TIMP1, and (F) TIMP2 released into the culture medium were measured by an ELISA assays as described in the Material and Methods section. The results are the mean from 7 donors. The data represent the mean ± SEM. An * indicates a p value of < 0.05.</p

Telomerase activity.

<p>A comparison of the relative telomerase activities for (A) SVF and ADSCs at a specific time and date (7,14,21 and 30 days) and (B) through the passages (P1-P4). One million cells were re-suspended in 1×Lysis Buffer, and then incubated on ice for 30 min. The telomerase activity was detected by qPCR as indicated, in the Materials and Methods section. The results are from 10 donors. The data represent the mean ± SEM.</p

Aldehyde dehydrogenase activity.

<p>One million cells were separated in two tubes; 5 μl of a 1.5 mM DEAB stock solution was added to the “control” and 5 μl of activated ALDEFLUOR^® Substrate per mL of sample was added to the “test” tube. Cells were then incubated for 30 min in a 37°C. Finally, the tubes were analyzed by a flow cytometer. (A) A representative flow cytometry analysis of the ALDH activity; and (B) the average of ALDHbr cells results from 10 donors. The data represent the mean + SEM.</p

Differentiation capacity of adipose-derived cells.

<p>Cells at P1 and P4 were incubated for three weeks with adipogenic, osteogenic, and chondrogenic media. Representative images (B, C) of the intracellular lipid droplets that were confirmed by Oil Red O staining compared to (A) the control cells. (E, F) The presence of calcium deposit was visualized by Alizarin Red staining compared (D) to the control. (H, I) The presence of GAG was confirmed with Alcian Blue staining and (K, L) the solid chondrogenic micromass compared to the control (G, J).</p

Morphology, immunophenotype and viability of adipose-derived cells.

<p>The morphology, viability and immunophenotype of adipose-derived cells during passaging. ADSCs have fibroblastic features at P1, and P4. (A) The cell viability was confirmed by flow cytometry analysis. (B) The viability of ADSCs. (C) The flow cytometry histograms for a representative donor are displayed at passage 4 (P4). The percentage of cells stained positive is indicated in the upper right corner of each panel. The green line indicates the positively stained cells, whereas the purple line indicates the isotype-matched monoclonal antibody control.</p