El Atat Oula, Antonios Diane, Hilal George, Hokayem Nabil, Abou-Ghoch Joelle, Hashim Hussein, Serhal Rim, Hebbo Clara, Moussa Mayssam, Alaaeddine Nada
Regenerative Medicine and Inflammation Laboratory, Faculty of Medicine, St. Joseph University, Beirut, Lebanon.
Toxicology Laboratory, Faculty of Pharmacy, St. Joseph University, Beirut, Lebanon.
PLoS One. 2016 Sep 15;11(9):e0162332. doi: 10.1371/journal.pone.0162332. eCollection 2016.
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.
由于脂肪来源干细胞（ADSC）具有可塑性、分化能力以及旁分泌和营养作用，其在再生医学中的应用正在增加。尽管从脂肪组织中可获得大量细胞，但对于许多疾病或美容手术的治疗目的而言，这些细胞通常还不够。因此，需要在体外培养和扩增细胞数周。我们的目的是研究最少传代次数的长期增殖是否会影响ADSC的干性、旁分泌分泌和致癌标志物。通过流式细胞术观察初始基质血管成分（SVF）和连续传代的ADSC的免疫表型特性和醛脱氢酶（ALDH）活性。同时，通过q-PCR评估端粒酶活性以及癌基因和肿瘤抑制基因的相对表达。我们还通过ELISA评估传代ADSC的细胞因子分泌谱。扩增后的ADSC保留了其形态和表型特征。这些细胞在培养中维持长达12周直至P4，具有稳定的端粒酶和ALDH活性，且无TP53突变。此外，TP53、RB和MDM2的相对表达水平未受影响，而c-Myc的相对表达显著下降。最后，PGE2、STC1和TIMP2的分泌水平未受影响，但在P2时IL-6、VEGF和TIMP 1的水平显著下降。我们的结果表明，传代ADSC的扩增不会影响干细胞的分化能力，也不会使细胞在体外获得癌状态或致癌能力。
