The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells

Abstract

There has been an increasing interest in understanding how the mechanical properties of the microenvironment influence stem cell fate. We describe studies of the proliferation and differentiation of neural stem cells (NSCs) encapsulated within three-dimensional scaffolds--alginate hydrogels--whose elastic moduli were varied over two orders of magnitude. The rate of proliferation of neural stem cells decreased with increase in the modulus of the hydrogels. Moreover, we observed the greatest enhancement in expression of the neuronal marker beta-tubulin III within the softest hydrogels, which had an elastic modulus comparable to that of brain tissues. To our knowledge, this work represents the first demonstration of the influence of modulus on NSC differentiation in three-dimensional scaffolds. Three-dimensional scaffolds that control stem cell fate would be broadly useful for applications in regenerative medicine and tissue engineering.

Fig 1

Characterization of modulus of alginate hydrogels by rheological measurements: a) Elastic modulus of different alginate hydrogels during frequency sweep analysis - 0.25-10 (▲); 0.25-50 (■); 0.25-100 (●); 1-100 (◆); b): Viscous modulus of different alginate hydrogels during frequency sweep analysis - 0.25-10 (Δ); 0.25-50 (□); 0.25-100(○); 1-100(◊).

Fig 2

Influence of composition of alginate hydrogels on the elastic modulus (G′) at 8 Hz. Error bars represent the standard deviation.

Fig 3

Proliferation of NSCs after 7 days in alginate hydrogels having different values of the modulus. Error bars represent the standard deviation; p<0.01 based on Student's t-test.

Fig 4

Confocal images showing nestin (i) and β-tubulin III (ii) staining of NSCs grown inside alginate hydrogels having different values of the modulus (A: 0.25-10 (183 Pa); B: 0.25-50 (1028 Pa); C: 0.25-100 (1735 Pa); D: 1-100 (19700 Pa); E: Control) in medium containing 20 ng/mL FGF.

Fig 5

QRT-PCR results for β-tubulin III after 7 days of growth in alginate hydrogels having different values of the elastic modulus. The data reported represent the ratio of the expression of β-tubulin III and 18S ribosomal RNA normalized relative to the ratio in cells cultured in highest modulus hydrogel (1-100). Error bars represent the standard deviation; *p<0.0005, **p<0.05 compared with highest modulus hydrogel (1-100) based on Student's t-test.