Swiss Paraplegic Research, 6207, Nottwil, Switzerland.
Tissue Engineering for Orthopaedics and Mechanobiology (TOM), Department for Biomedical Research (DBMR), University of Bern, 3008, Bern, Switzerland.
Sci Rep. 2020 Nov 5;10(1):19084. doi: 10.1038/s41598-020-76202-2.
Mesenchymal stromal cells (MSC) are used in cell therapy, but results depend on the unknown quality of cell populations. Extended culture time of MSC increases their senescent levels, leading to a critical loss of cell fitness. Here, we tested the suitability of MSC-sorting based on their FACS autofluorescence profile, for a rapid and non-invasive method of senescent cell elimination. Cells were classified in low- (LA) and high- (HA) autofluorescence groups, and results compared to the original MSC population (control). Three days after sorting, cells were screened by replicative senescence markers (cell volume, SA-β-Gal assay and gene/protein expression) and MSC differentiation assays. The transcriptional profiles of sorted MSC were also analyzed by RNA-Seq. Compared to control, LA cells had 10% lower cell volume and autofluorescence, and 50% less SA-β-Gal + cells. Instead, HA cells had 20% higher cell volume and autofluorescence, and 120% more SA-β-Gal + cells. No changes in replicative senescence and differentiation potentials were observed between all groups. However, 68 genes (16 related to senescence) were significantly differentially expressed (DEG) between LA and other groups. Biological network of DEG identified CXCL12 as topological bottleneck. In summary, MSC sorting may have practical clinical implications to enhance the results of MSC-based therapies.
间充质基质细胞 (MSC) 被用于细胞治疗，但结果取决于细胞群体未知的质量。MSC 的延长培养时间会增加其衰老水平，导致细胞适应性的严重丧失。在这里，我们测试了基于 FACS 自发荧光特征的 MSC 分选，作为一种快速、非侵入性的衰老细胞去除方法的适用性。将细胞分为低自发荧光 (LA) 和高自发荧光 (HA) 组，并将结果与原始 MSC 群体（对照）进行比较。分选后 3 天，通过复制性衰老标志物（细胞体积、SA-β-Gal 测定和基因/蛋白表达）和 MSC 分化测定筛选细胞。还通过 RNA-Seq 分析了分选 MSC 的转录谱。与对照相比，LA 细胞的细胞体积和自发荧光降低了 10%，SA-β-Gal+细胞减少了 50%。相反，HA 细胞的细胞体积和自发荧光增加了 20%，SA-β-Gal+细胞增加了 120%。在所有组之间未观察到复制性衰老和分化潜能的变化。然而，68 个基因（16 个与衰老相关）在 LA 和其他组之间存在显著差异表达（DEG）。DEG 的生物学网络确定了 CXCL12 作为拓扑瓶颈。总之，MSC 分选可能具有实际的临床意义，可以提高基于 MSC 的治疗效果。
