Human bone marrow-derived mesenchymal stromal cells (MSCs) obtained from aged patients are prone to senesce and diminish their differentiation potential, therefore limiting their usefulness for osteochondral regenerative medicine approaches or to study age-related diseases, such as osteoarthiritis (OA). MSCs can be transduced with immortalizing genes to overcome this limitation, but transduction of primary slow-dividing cells has proven to be challenging. Methods for enhancing transduction efficiency (such as spinoculation, chemical adjuvants, or transgene expression inductors) can be used, but several parameters must be adapted for each transduction system. In order to develop a transduction method suitable for the immortalization of MSCs from aged donors, we used a spinoculation method. Incubation parameters of packaging cells, speed and time of centrifugation, and valproic acid concentration to induce transgene expression have been adjusted. In this way, four immortalized MSC lines (iMSC#6, iMSC#8, iMSC#9, and iMSC#10) were generated. These immortalized MSCs (iMSCs) were capable of bypassing senescence and proliferating at a higher rate than primary MSCs. Characterization of iMSCs showed that these cells kept the expression of mesenchymal surface markers and were able to differentiate towards osteoblasts, adipocytes, and chondrocytes. Nevertheless, alterations in the CD105 expression and a switch of cell fate-commitment towards the osteogenic lineage have been noticed. In conclusion, the developed transduction method is suitable for the immortalization of MSCs derived from aged donors. The generated iMSC lines maintain essential mesenchymal features and are expected to be useful tools for the bone and cartilage regenerative medicine research.

中文翻译：

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永生的供体永生化间质基质细胞，同时保持其基本功能。

从老年患者获得的人骨髓源间充质基质细胞（MSC）易于衰老并降低其分化潜能，因此限制了它们在骨软骨再生医学方法或研究与年龄相关的疾病（例如骨关节炎）中的用途。可以用永生化基因转导MSC，以克服这一限制，但事实证明，慢速分化原代细胞的转导具有挑战性。可以使用提高转导效率的方法（例如旋转接种，化学佐剂或转基因表达诱导剂），但是必须为每个转导系统调整几个参数。为了开发一种适用于永生供体的MSC永生化的转导方法，我们使用了旋转接种法。包装细胞的孵育参数 调整了离心速度和时间，以及丙戊酸浓度以诱导转基因表达。这样，生成了四个永生化的MSC线（iMSC＃6，iMSC＃8，iMSC＃9和iMSC＃10）。这些永生化的MSC（iMSC）能够绕过衰老并以比原代MSC更高的速率增殖。iMSC的表征表明，这些细胞保持了间充质表面标志物的表达，并且能够分化为成骨细胞，脂肪细胞和软骨细胞。然而，已经注意到CD105表达的改变和细胞命运承诺向成骨谱系的转变。总之，开发的转导方法适用于永生供体来源的MSC的永生化。