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

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

Cell yield and the population doubling time (PDT) of passaged ADSCs (n = 6).

<p>Cell yield and the population doubling time (PDT) of passaged ADSCs (n = 6).</p

List of primer sequences.

<p>List of primer sequences.</p

TP53 sequencing and the relative expression of the tumor suppressor genes TP53 and RB and the oncogenes c-Myc, MDM2, and hTERT.

<p>(A) The PCR products of TP53 exon 2 to 11 from passage 0 and 4 ADSCs. (B) BLAST (Basic Local Alignment Search Tool) was used to compare the TP53 sequence to the wild type TP53 sequence. This figure from one donor is a representative for all donors. (C) Relative expression of the tumor suppressor genes and oncogenes. Total RNA was purified and the cDNA was prepared from total RNA using an iScript kit. The relative expression levels of the tumor suppressor genes TP53 and RB, and the expression levels of the oncogenes c-Myc, MDM2, and hTERT from ADSCs at P0 and P4 was performed using quantitative PCR with specific primers as indicated in the Material and Methods section. The results are the mean from 5 donors. The data were normalized to GAPDH and presented as the mean ± SEM. An * indicates a p value of < 0.05.</p

List of primer sequences for real-time PCR.

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

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