Development of an integrin α_v-based universal marker, capable of both prediction and direction of stem cell fate

Integrin-mediated focal adhesion (FA) and subsequent cytoskeletal reorganization influence cell morphology, migration, and ultimately cell fate. Previous studies have used various patterned surfaces with defined macroscopic cell shapes or nanoscopic FA distributions to explore how different substrates affect the fate of human bone marrow mesenchymal stem cells (BMSCs). However, there is currently no straightforward relationship between BMSC cell fates induced by patterned surfaces and FA distribution substrates. In this study, we conducted single-cell image analysis of integrin α_v-mediated FA and cell morphological features of BMSCs during biochemically induced differentiation. This enabled the identification of distinct FA features that can discriminate between osteogenic and adipogenic differentiation, demonstrating that integrin α_v-mediated focal adhesion (FA) can be used as a non-invasive biomarker for real time observation. Based on these results, we developed an organized microscale fibronectin (FN) patterned surface where the fate of BMSC could be precisely manipulated by these FA features. Notably, even in the absence of any biochemical inducers, such as those contained in the differentiation medium, BMSCs cultured on these FN patterned surfaces exhibited upregulation of differentiation markers comparable to BMSCs cultured using conventional differentiation methods. Therefore, the present study reveals the application of these FA features as universal markers not only for predicting differentiation status, but also for regulating cell fate by precisely controlling the FA features with a new cell culture platform. STATEMENT OF SIGNIFICANCE: Although the effects of material physiochemical properties on cell morphology and subsequent cell fate decisions have been extensively studied, a simple yet intuitive correlation between cellular features and differentiation remains unavailable. We present a single cell image-based strategy for predicting and directing stem cell fate. By using a specific integrin isoform, integrin α_v, we identified distinct geometric features that can be used as a marker for discriminating between osteogenic and adipogenic differentiation in real-time. From these data, new cell culture platforms capable of regulating cell fate by precisely controlling FA features and cell area can be developed.