Abstract
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.
Highlights
Regenerative medicine is an exciting new field in which different techniques are used to mend damaged organs and tissues
To characterize our primary cells and cells at different passages (P0-passage 4 (P4)), flow cytometry analysis was performed on the stromal vascular fraction (SVF) and passaged adipose-derived stem cells (ADSC)
The seeded cells were purified by changing the media and subculturing to become ADSCs, which were adherent to the plastic and showed a fibroblast-like morphology (Fig 2A)
Summary
Regenerative medicine is an exciting new field in which different techniques are used to mend damaged organs and tissues. Adult mesenchymal stem cells represent an attractive candidate for tissue regeneration and repair because they have low immunogenicity, are non-tumorigenic and are not subject to any ethical issues. The International Society of Cellular Therapy (ISCT) proposed the minimum criteria for defining human mesenchymal stem cells (MSCs) These cells are plastic adherent and have a fibroblast-like morphology. They must express CD73, CD90, and CD105, but lack the expression of CD34, CD45, CD14 or CD11b, CD79α or CD19, class II major histocompatibility complex (MHCII) molecule (mainly HLA-DR) and co-stimulatory molecules such as B7-1, B7-2, CD80, CD86, CD40 and CD40L. They must be able to differentiate in vitro into mesodermal cellular lineages, adipocytes, osteoblasts, and chondrocytes [1,2,3,4]
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