The interplay of peptide affinity and scaffold stiffness on neuronal differentiation of neural stem cells

Abstract

Cells are sensitive to physical cues in their environment, such as the stiffness of the substrate, peptide density, and peptide affinity. Understanding how neural stem cells (NSCs) sense and respond to these matrix cues has the potential to improve disease outcome, particularly if a regenerative response can be exploited. While the material properties are known to influence other stem cells, little is known about how NSC differentiation is altered by this interplay of mechanical, or bulk properties, with peptide concentration and affinity, or microscale properties. We are interested in the combined effect of bulk and microscale features in an in vitro hydrogel model and therefore we investigated NSC differentiation by focusing on integrin interactions via RGD peptide affinity and concentration. Our studies demonstrated that the peptide concentration affected adhesion as there were more cells on scaffolds with 1 mM RGD than 2.5 mM RGD. The hydrogel stiffness affected neurite length in differentiating NSCs, as 0.1-0.8 kPa substrates promoted greater neurite extension than 4.2-7.9 kPa substrates. The NSCs differentiated towards β-ΙΙΙ tubulin positive cells on scaffolds with RGD after 7 days and those scaffolds containing 1 mM linear or cyclic RGD had longer neurite extensions than scaffolds containing 0.1 or 2.5 mM RGD. While peptide affinity had a lesser effect on the NSC response in our hydrogel system, blocking actin, myosin II, or integrin interactions resulted in changes to the cell morphology and focal adhesion assembly. Overall, these results demonstrated NSCs are more responsive to a change in tissue stiffness than peptide affinity in the range of gels tested, which may influence design of materials for neural tissue engineering.

Soft Matrix Combined With BMPR Inhibition Regulates Neurogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells

“…Stem cells are extremely sensitive to their physical environment, but it is not clear how physical stimuli signals are transduced in the cells (Stukel and Willits, 2018). It has been reported that the BMP signaling pathway is a crucial pathway associated with neural induction of stem cells on stiff surfaces, tissue development, and cell differentiation (Thompson and Chan, 2016).…”

Hydrogels Derivatized With Cationic Moieties or Functional Peptides as Efficient Supports for Neural Stem Cells

“…The adult NSCs proliferated and differentiated on all gels with a modulus greater than 100 Pa and a peak level of the neuronal marker β-III tubulin was observed on gels with a modulus of around 500 Pa which resembles the stiffness of brain tissue. Also the neurite outgrowth was increased in hydrogels with a similar stiffness of 400 Pa in a HA based RGD-modified hydrogel (Tarus et al, 2016) and in a PEGDA-RGD-based hydrogels the neurite outgrowth was increased at a stiffness of 0.1 and 0.8 kPa, which were the lowest consistence tested (Stukel and Willits, 2018). Together this indicates that neurites preferentially grow toward an environment that is less stiff (Long and Huttner, 2019).…”

“…Together this indicates that neurites preferentially grow toward an environment that is less stiff (Long and Huttner, 2019). Additionally Stukel and Willits (2018) carried out adhesion studies with the 2-D modified PEGDA hydrogels and could show that the concentration of the peptide and the cell density are important for the adhesion effect of RGD. More precisely they exposed that a high concentration of 2.5 mM RGD decreased the adhesion at a cell density of 50 × 10 3 cells cm −2 , whereas a concentration of 0.1 and 1 mM, as well as a lower cell density of 10 × 10 3 cm −2 , exhibited a better result.…”

“…The best results regarding migration and formation of neurons were obtained from gels with an elastic modulus of 3800 to 5300 Pa and a lateral concentration of 0.32-0.60 µmol mg −1 . While these results already show that hydrogels containing cationic charges are promising tools to influence the behavior of NSCs, many researchers focus their interest on hydrogels with peptide sequences derived from natural proteins from the ECM (Sun et al, 2017;Stukel and Willits, 2018).…”

Regenerative Medicine Therapies for Targeting Neuroinflammation After Stroke

“…If the pore size of a hydrogel is too low then nutrients and oxygen within media may not efficiently permeate through the entire structure, or could cause a concentration gradient; potentially leading to necrotic regions ( 102 , 103 ). The stiffness of a hydrogel regulates the phenotype of cells, with mechanical interactions between cells of the NVU and the ECM through hydrostatic pressures ( 104 ) and CAP binding ( 105 , 106 ), even directing the differentiation of stem cells ( 105 – 108 ). Crosslinking is the mechanism by which a pre-gelation hydrogel becomes solid, with the initiation of inter-molecular physical or chemical bonds maintaining a 3D structure.…”