Cell and Developmental Biology graduate program in Molecular Biosciences, Rutgers University, Piscataway, NJ, 08854, USA.
Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, 08854, USA.
Sci Rep. 2019 Jul 17;9(1):10377. doi: 10.1038/s41598-019-46682-y.
This study describes a new approach to discern early divergence in stem cell lineage progression via temporal dynamics of the cytoskeletal protein, F-actin. The approach involves real-time labeling of human mesenchymal stem cells (MSCs) and longitudinal tracking of the turnover dynamics of a fluorogenic F-actin specific probe, SiR-actin (SA). Cells cultured in media with distinct lineage factors and labeled with SA showed lineage specific reduction in the actin turnover shortly after adipogenic (few minutes) and chondrogenic (3-4 hours) commitment in contrast to osteogenic and basal cultured conditions. Next, composite staining of SA along with the competing F-actin specific fluorescent conjugate, phalloidin, and high-content image analysis of the complementary labels showed clear phenotypic parsing of the sub-populations as early as 1-hour post-induction across all three lineages. Lastly, the potential of SA-based actin turnover analysis to distinguish cellular aging was explored. In-vitro aged cells were found to have reduced actin turnover within 1-hour of simultaneous analysis in comparison to cells of earlier passage. In summary, SiR-actin fluorescent reporter imaging offers a new platform to sensitively monitor emergent lineage phenotypes during differentiation and aging and resolve some of the earliest evident differences in actin turnover dynamics.
本研究描述了一种通过细胞骨架蛋白 F-actin 的时间动态来辨别干细胞谱系进展早期分歧的新方法。该方法涉及实时标记人间充质干细胞（MSCs）和荧光 F-actin 特异性探针 SiR-actin（SA）的周转率动力学的纵向跟踪。在具有不同谱系因子的培养基中培养并标记 SA 的细胞在成脂（数分钟）和软骨（3-4 小时）分化后不久表现出与成骨和基础培养条件相比，细胞骨架周转率明显降低。接下来，SA 与竞争的 F-actin 特异性荧光缀合物鬼笔环肽的复合染色以及互补标记物的高内涵图像分析表明，早在诱导后 1 小时，所有三种谱系都可以清晰地区分表型亚群。最后，还探索了基于 SA 的肌动蛋白周转率分析来区分细胞衰老的潜力。与早期传代细胞相比，体外衰老细胞在同时分析后 1 小时内发现肌动蛋白周转率降低。总之，SiR-actin 荧光报告基因成像为在分化和衰老过程中灵敏监测新兴的谱系表型提供了一个新平台，并解决了肌动蛋白周转率动力学中一些最早的明显差异。
