College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea.
College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea.
Biomed Res Int. 2019 May 21;2019:3093545. doi: 10.1155/2019/3093545. eCollection 2019.
Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.
长期在定义的培养条件下扩增间充质干细胞（MSCs）是人类干细胞治疗所必需的。然而，这会改变 MSCs 的特征。由于实时定量聚合酶链反应（qRT-PCR）被广泛用作比较特征的关键分析方法之一，因此在每种实验条件下验证参考基因（RGs）的归一化对于获得可靠的 qRT-PCR 结果非常重要。因此，评估了在定义的培养条件下长期扩增（多达 20 代）骨髓和脐血来源的间充质干细胞（BM-MSCs 和 UCB-MSCs）的最稳定的 RG。在长期扩增过程中，BM-MSCs 比 UCB-MSCs 表现出更明显的特征改变，如分化能力、增殖、衰老和 RG 的表达。RG 验证程序（GeNorm 和 NormFinder）表明，无论 MSC 类型和长期培养扩增如何，PPIA、HPRT1 和 YWHAZ 均适合 qRT-PCR 中的归一化，而传统 RG（ACTB 和 GAPDH）在长期扩增的 MSC 中稳定性较差。此外，在长期扩增的 BM-MSCs 中使用这些 RG 对 OCT4 表达进行归一化的结果表明，稳定性较差的 RG（GAPDH）与其他 RG 相比显示出相反的数据。因此，强烈建议在长期扩增的 MSC 中使用 PPIA、HPRT1 和 YWHAZ 等 RG 进行 qRT-PCR 实验的归一化，以生成准确可靠的数据。
