Enrichment and Identification of Neural Stem Cells in Neurospheres Using Rigidity-Tunable Gels

Abstract

Stem cell fate specification is known to be regulated by mechanical cues such as cell–cell interaction and the rigidity of the extracellular matrix (ECM). At present, spheroid cultures of stem cells are conventional starting materials to generate organoids with tissue-like structures. However, the influence of ECM rigidity on stem cell mechanobiology has only been briefly explored in in vitro three-dimensional (3D) cultures, which presents better physiological relevance than monolayer cultures. To investigate how ECM rigidity acts on neural stem cells (NSCs) to drive specific biological processes, we performed live 3D-imaging on neurospheres in medium, in 1% and 1.5% agarose gel-medium matrix. In this study, we also report a novel method of 3D spheroid expansion stress quantification. Our results revealed that neurospheres in stiffer ECM exhibited higher stem cell ratio than those in medium. Moreover, we observed a negative correlation between the signal intensity of the NSC marker, SSEA-1 (CD15), and mean stresses exerted by the cell. This observation is implicative of a sparse actomyosin network in NSCs than nonstem cells. Based on these results, we propose that enrichment of NSCs or directed differentiation may be carried out by tailoring ECM rigidity and possibly its composition.

Impact Statement

Neural stem cells (NCSs) are integral to establishing in vitro models and regenerative medicine. To this day, there is an unmet need to enrich these cells from a heterogeneous cell population for clinical applications without irreversible manipulation. We identified a method to propagate human NCSs via computational analysis of their mechanical signature. In this study, we report a novel analytical method for mechanical forces in three-dimensional cultures. Further, our results revealed that stemness may, in part, be mediated by physical properties of the extracellular matrix. In conclusion, our findings have potential implications in understanding stem cell mechanobiology for enrichment or differentiation.