Ravid Orly, Shoshani Ofer, Sela Meirav, Weinstock Ada, Sadan Tommy Weiss, Gur Eyal, Zipori Dov, Shani Nir
Stem Cell Res Ther. 2014 Dec 17;5(6):139. doi: 10.1186/scrt529.
Mesenchymal 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.
MSCs 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).
We 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.
The 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.
间充质干细胞（MSC）具有多能性，可来源于多种组织。尽管MSC具有许多基本特征，但它们通常表现出细微的组织特异性差异。我们之前证明，骨髓（BM）来源的MSC在培养过程中经常会变成多倍体。这种趋势是由从二倍体状态转变为多倍体状态期间H19长链非编码RNA表达的降低所介导的。
从小鼠的骨髓和脂肪组织中分离出MSC，并在常氧和低氧培养条件下进行扩增。用碘化丙啶对细胞进行染色，并通过流式细胞术评估其倍性。通过定量实时PCR比较独立的MSC制剂的基因表达，并通过蛋白质印迹分析比较蛋白质表达水平。通过特异性小发夹RNA（shRNA）使MSC中的p53沉默。
我们着手研究基因组不稳定性是否是源自不同组织的MSC所共有的。结果表明，脂肪来源的MSC（ASC）在培养过程中倾向于保持二倍体状态，而绝大多数BM来源的MSC会变成多倍体。与BM来源的MSC相比，ASC的二倍体表型与H19表达降低相关。在低氧条件下（3%氧气），ASC和BM来源的MSC均显示H19和血管内皮生长因子A的RNA表达增加。重要的是，在两种氧气条件下，ASC的基因表达变异性均明显低于BM来源的MSC，表明其具有更高的同质性。基因表达分析显示，通常由DNA损伤诱导的p53靶基因在基础条件下的ASC中上调。然而，与ASC相比，用DNA损伤剂处理后BM来源的MSC中p53的激活明显增强。我们发现p53参与维持ASC的稳定二倍体状态，因为p53 shRNA诱导了ASC中的倍性变化，但未诱导BM来源的MSC中的倍性变化。
小鼠ASC基因组稳定性的增加及其较低的H19表达和相对同质性表明，与BM来源的MSC相比，ASC具有组织特异性的更高稳定性，这可能是由于p53的活性更高。来自不同组织来源的MSC之间的组织特异性差异可能对各种MSC在体外和体内的应用产生重要影响。
