Relative genomic stability of adipose tissue derived mesenchymal stem cells: analysis of ploidy, H19 long non-coding RNA and p53 activity

Abstract

IntroductionMesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state.MethodsMSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA).ResultsWe set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs.ConclusionsThe increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt529) contains supplementary material, which is available to authorized users.

Identification and Characterization of Long Non-Coding RNAs in Osteogenic Differentiation of Human Adipose-Derived Stem Cells

“…Here, we found that H19 expression decreased significantly during osteogenic differentiation of human ASCs. To the best of our knowledge, only one study showed reduced expression of H19 in ASCs as compared with BMSCs [38]; however, no report exists regarding H19 expression during osteogenesis of ASCs. This discrepancy might be explained by evidence showing that H19 expression is regulated in a tissue- and cell-specific manner during embryogenesis [39].…”

The tissue specific nature of mesenchymal stem/stromal cells: gaining better understanding for improved clinical outcomes

“…In BM MSCs, polyploidy appeared to be a more stable state than diploidy as diploid BM MSCs were significantly more tumorigenic following subcutaneous administration, and expressed much higher levels of H19. The correlation between H19 expression and the tendency of BM MSCs to become polyploid was further substantiated in another recent publication that compared the genomic stability of BM MSCs to that of ASCs [18] . Altogether, it was found that 76% (13 out of 17 cell preparations) of mouse BM MSCs preparations, demonstrated a polyploid phenotype compared to only 9% polyploidy (3 out of 32 cell preparations) of ASCs.…”

“…The recent study [20] suggested that in some instances the opposite is true, namely tetraploidy is necessary for p53 wild type cells to remain non-tumorigenic. It was additionally found that the basal p53 activity, evident by the RNA expression of various p53 target genes, was significantly higher in ASCs compared to BM MSCs [18] . Interestingly, three different DNA damage stresses (UV eradiation, oxidative stress by H 2 0 2 and doxorubicin) all resulted in a significant p53 activation in both tetraploid BM MSCs and in ASCs demonstrating the presence of a wild type p53 in these cells.…”

“…Alternatively, the proposed signature may still be evident in the more heterogeneous but early progenitor population. In two recently published studies we therefore compared BM marrow derived MSCs to adipose derived MSCs (ASCs) [18] and abdominal ASCs (aASCs) to subcutaneous ASCs (scASCs) [19] . It was found that ASCs are more genetically stable than BM MSCs and demonstrate a more homogenous nature in their response to changing or stressful conditions.…”

“…Interestingly, three different DNA damage stresses (UV eradiation, oxidative stress by H 2 0 2 and doxorubicin) all resulted in a significant p53 activation in both tetraploid BM MSCs and in ASCs demonstrating the presence of a wild type p53 in these cells. It thus seems that the increased basal p53 activity together with the reduced H19 expression contributes to the superior ability of ASCs to remain diploid [18] . Importantly, a non-transformed state is achieved in different ways in ASCs and BM MSCs, as the former maintain a diploid genome whereas the latter become tetraploid.…”