High content image analysis of focal adhesion-dependent mechanosensitive stem cell differentiation

Abstract

Human mesenchymal stem cells (hMSCs) receive differentiation cues from a number of stimuli, including extracellular matrix (ECM) stiffness. The pathways used to sense stiffness and other physical cues are just now being understood and include proteins within focal adhesions. To rapidly advance the pace of discovery for novel mechanosensitive proteins, we employed a combination of in silico and high throughput in vitro methods to analyze 47 different focal adhesion proteins for cryptic kinase binding sites. High content imaging of hMSCs treated with small interfering RNAs for the top 6 candidate proteins showed novel effects on both osteogenic and myogenic differentiation; Vinculin and SORBS1 were necessary for stiffness-mediated myogenic and osteogenic differentiation, respectively. Both of these proteins bound to MAPK1 (also known as ERK2), suggesting that it plays a context-specific role in mechanosensing for each lineage; validation for these sites was performed. This high throughput system, while specifically built to analyze stiffness-mediated stem cell differentiation, can be expanded to other physical cues to more broadly assess mechanical signaling and increase the pace of sensor discovery.

Vinculin promotes nuclear localization of TAZ to inhibit ECM stiffness-dependent differentiation into adipocytes

“…Our previous research revealed that the interaction of vinculin with vinexin α functions as a mechanosensor for ECM stiffness, and this interaction is a prerequisite for the stiffness-dependent regulation of vinculin status and cell migration in mouse embryonic fibroblasts (Yamashita et al, 2014). In agreement with our findings, Holle et al reported that vinculin and vinexin have an essential role in myogenic differentiation (Holle et al, 2016) during the process of revising our manuscript. Thus, the present study extends our understanding of the vinculin function in mechanosensing and mechanotransduction to the regulation of cell differentiation and to MSCs.…”

Vinexin family (SORBS) proteins play different roles in stiffness-sensing and contractile force generation

“…A recent report has shown that vinexin-α and CAP are necessary for myogenic differentiation on 15 kPa gels and osteogenic differentiation on 42 kPa gels in human mesenchymal stem cells, respectively (Holle et al, 2016). In addition, vinculin is also necessary for stiffness-dependent myogenesis and, to a lesser extent, involved in osteogenesis (Holle et al, 2013(Holle et al, , 2016. We have also clarified that vinculin regulates stiffness-dependent adipogenesis of the mouse mesenchymal stem cell line ST2 cells (Kuroda et al, 2017).…”

“…These results suggest that CAP has a higher affinity for vinculin compared with vinexin-α, and that CAP forms a ternary complex with vinculin and F-actin more effectively than vinexin-α. A recent report has shown that vinexin-α and CAP are necessary for myogenic differentiation on 15 kPa gels and osteogenic differentiation on 42 kPa gels in human mesenchymal stem cells, respectively (Holle et al, 2016). In addition, vinculin is also necessary for stiffness-dependent myogenesis and, to a lesser extent, involved in osteogenesis (Holle et al, 2013(Holle et al, , 2016.…”

Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels

“…Physiological interfaces, such as those at neuromuscular junctions, and pathological boundaries, e.g., infarcted fibrous heart tissue adjacent to healthy myocardium, are prevalent in vivo and imply that mechanical cues not only help guide differentiation but play critical regulatory roles in disease response. Recent studies have identified substrate stiffness as a significant factor in cell spreading (7,8), migration (9), proliferation (10), and differentiation (11,12) using both adipose-derived (13) and bone marrow-derived stem cells (14). When mimicking the stiffness of neural (∼1 kPa), muscle (∼12 kPa), and bone (∼30 kPa) tissues (15,16), substrate stiffness can induce differentiation toward those specific tissue types.…”