The effect of Young’s modulus on the neuronal differentiation of mouse embryonic stem cells

Shahzad Ali,Ivan B Wall,Chris Mason,Andrew E Pelling,Farlan S Veraitch

doi:10.1016/j.actbio.2015.07.008

Abstract

There is substantial evidence that cells produce a diverse response to changes in ECM stiffness depending on their identity. Our aim was to understand how stiffness impacts neuronal differentiation of embryonic stem cells (ESC’s), and how this varies at three specific stages of the differentiation process. In this investigation, three effects of stiffness on cells were considered; attachment, expansion and phenotypic changes during differentiation. Stiffness was varied from 2kPa to 18kPa to finally 35kPa. Attachment was found to decrease with increasing stiffness for both ESC’s (with a 95% decrease on 35kPa compared to 2kPa) and neural precursors (with a 83% decrease on 35kPa). The attachment of immature neurons was unaffected by stiffness. Expansion was independent of stiffness for all cell types, implying that the proliferation of cells during this differentiation process was independent of Young’s modulus. Stiffness had no effect upon phenotypic changes during differentiation for mESC’s and neural precursors. 2kPa increased the proportion of cells that differentiated from immature into mature neurons. Taken together our findings imply that the impact of Young’s modulus on attachment diminishes as neuronal cells become more mature. Conversely, the impact of Young’s modulus on changes in phenotype increased as cells became more mature.

Highlights

Embryonic stem (ES) cells, derived from the early mammalian embryo are unique in their ability to both self renew indefinitely in vitro [1] as well as differentiate into cells from any of the 3 germ layers and form any tissue within the body [2]
On the day of passaging, cells were washed with phosphate buffer saline (PBS) and trypsinised by incubating with trypsin for 3 min before being resuspended in fresh medium and plated onto fresh gelatin-coated tissue culture polystyrene (TCP) flasks
Three GXG substrates were synthesised by varying gelatin concentration in water or PBS for a given concentration of glutaraldehyde

Summary

Introduction

Embryonic stem (ES) cells, derived from the early mammalian embryo are unique in their ability to both self renew indefinitely in vitro [1] as well as differentiate into cells from any of the 3 germ layers (endoderm, mesoderm and ectoderm) and form any tissue within the body [2]. As a result of these two properties, there has been much interest generated in potential therapeutic applications for embryonic stem cells, including treatments for Parkinson’s disease [3,4,5,6], diabetes [7,8,9] and cardiovascular disease [10,11,12,13,14]. One approach to improve the efficiency of stem cell processes has been to investigate the role of the mechanical microenvironment. This includes factors such as cyclic strain [17], shear from fluid flow [18] and extracellular matrix elasticity [19,20,21] which have all been shown to be key regulators of stem cell differentiation. Culturing cells on a matrix elasticity similar to that of the

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