In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery, and increased activity of the senescence-associated β-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac™ Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.

在过去的几十年里，科学界不遗余力地阐明间充质基质细胞 (MSCs) 的治疗潜力。不幸的是，考虑到这些细胞在再生医学领域的未来治疗应用，体外细胞衰老伴随着增殖能力的丧失是一个主要缺点。尽管对人类医学中复制性衰老机制的深入了解已经发生了巨大变化，但关于犬 MSCs 复制性衰老的知识仍然很少。因此，我们开发了一种高内涵分析工作流程，以同时研究犬脂肪来源的 MSC (cAD-MSC) 衰老的三个重要特征：形态变化、细胞周期阻滞机制的激活和衰老相关 β 的活性增加-半乳糖苷酶。我们利用这个工具来证明 cAD-MSC 的传代导致出现衰老表型和增殖停滞。使用新设计的 PiggyBac™ 转座子系统使这些细胞永生化后，这在一定程度上得到了防止，该系统允许人类多梳环指原癌基因 BMI1 和人类端粒酶逆转录酶在相同的启动子下表达。我们的结果表明，与未经处理的 cAD-MSCs 相比，用这种新载体永生化的 cAD-MSCs 保持其增殖能力和分化潜能的时间更长。本研究不仅提供了一种通过高内涵分析方法研究真核细胞复制性衰老的工作流程，而且为快速有效地生成永生化 MSC 系铺平了道路。