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Issue 10, 2016
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High content image analysis of focal adhesion-dependent mechanosensitive stem cell differentiation

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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.

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

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Supplementary files

Article information


Submitted
04 May 2016
Accepted
29 Aug 2016
First published
30 Aug 2016

Integr. Biol., 2016,8, 1049-1058
Article type
Paper
Author version available

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