Mechanics regulates fate decisions of human embryonic stem cells

Abstract

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.

Figure 1. Differential cytoskeleton contractility and FA distribution for single Oct⁺ and Oct⁻ hESCs.

(A) Quantification of subcellular traction forces for single Oct⁺ (top row) and Oct⁻ (bottom row) hESCs using the PDMS micropost array. (B&C) Bar plots of total traction forces per cell (B) and traction force per cell area (C) for both single Oct⁺ and Oct⁻ hESCs. Data represents the means ± s.e.m from 3 independent experiments. **, p<0.01. (D) Immunofluorescence images showing FA distributions in single hESCs (left: Oct⁺; right: Oct⁻), as indicated by vinculin staining. Scale bar, 20 µm.

Figure 2. Matrix mechanic-mediated behaviors of single hESCs on PDMS micropost arrays with different rigidities.

(A) Bar plot of percentage of Oct⁺ cells for single hESCs plated on the PDMS micropost arrays with different rigidities. (B&C) Traction force per cell area (B) and total traction forces per cell (C) for both Oct⁺ and Oct⁻ cells as a function of the PDMS micropost array rigidity. (D) Phase contrast and immunofluorescence images of hESCs treated with or without blebbistatin on both soft (E_eff = 1.92 kPa) and rigid (E_eff = 1,218.4 kPa) PDMS micropost arrays. Scale bar, 50 µm. (E) Bar plot of percentage of Oct⁺ cells for blebbistatin treated hESCs and untreated controls as a function of the PDMS micropost array rigidity. Data in E was normalized to the value for untreated hESCs plated on the rigid micropost array under the 24-hr treatment condition. Data in A–C and E represents the means ± s.e.m from 3 independent experiments. *: p<0.05; **: p<0.01; NS: p>0.05.

Figure 3. Matrix mechanics-mediated cellular functions of small aggregates of hESCs on PDMS micropost arrays with different rigidities.

(A) Bar plots of percentage of Oct⁺ cells for clustered hESCs of different sizes as a function of the PDMS micropost rigidity. (B) Plot of average total traction force per cell for both Oct⁺ and Oct⁻ cells contained in different sized hESC aggregates. Data in A & B represents the means ± s.e.m from 3 independent experiments. *: p<0.05; **: p<0.01; NS: p>0.05. (C) Immunofluorescence images showing FA distributions in Oct⁺ (top) and Oct⁻ (bottom) hESC aggregates, as indicated by vinculin staining. Scale bar, 25 µm.

Figure 4. E-cadherin expression of hESCs modulated by substrate rigidity.

(A) Immunofluorescence images taken for undifferentiated (Oct⁺) and differentiated (Oct⁻) hESC colonies on soft (E_eff = 1.92 kPa) and rigid (E_eff = 1,218.4 kPa) PDMS micropost arrays, as indicated. Differentiated hESC colonies were marked with an arrow. Scale bars, 50 µm. (B) Phase contrast and immunofluorescence images of hESCs treated with or without DECMA-1 on both soft (E_eff = 1.92 kPa) and rigid (E_eff = 1,218.4 kPa) PDMS micropost arrays. Scale bar, 50 µm. (C) Bar plot of percentage of Oct⁺ cells for DECMA-1 treated hESCs and untreated controls as a function of the PDMS micropost rigidity. Data represents the means ± s.e.m from 3 independent experiments. *: p<0.05; **: p<0.01.