Fluorescence-based actin turnover dynamics of stem cells as a profiling method for stem cell functional evolution, heterogeneity and phenotypic lineage parsing.

Stem cells are widely explored in regenerative medicine as a source to produce diverse cell types. Despite the wide usage of stem cells like mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), there is a lack of robust methods to rapidly discern the phenotypic and functional heterogeneity of stem cells. The organization of actin cytoskeleton has been previously used to discern divergent stem cell differentiation pathways. In this paper, we highlight the versatility of a cell profiling method for actin turnover dynamics. Actin filaments in live stem cells are labeled using SiR-actin, a cell permeable fluorogenic probe, to determine the endogenous actin turnover. Live MSC imaging after days of induction successfully demonstrated lineage specific change in actin turnover. Next, we highlighted the differences in the cellular heterogeneity of actin dynamics during adipogenic or osteogenic MSC differentiation. Next, we applied the method to differentiating iPSCs in culture, and detected a progressive slowdown in actin turnover during differentiation upon stimulation with neural or cardiac media. Finally, as a proof of concept, the actin dynamic profiling was used to isolate MSCs via flow cytometry prior to sorting into three distinct sub-populations with low, intermediate or high actin dynamics. A greater fraction of MSCs with more rapid actin dynamics demonstrated increased inclination for adipogenesis, whereas, slower actin dynamics correlated with increased osteogenesis. Together, these results show that actin turnover can serve as a versatile biomarker to not only track cellular phenotypic heterogeneity but also harvest live cells with potential for differential phenotypic fates.