Directed Neuronal Differentiation of SH-SY5Y Human Neuroblastoma Cells on 2D Matrices Containing Recombinant Spidroins Modified with Cell Adhesion Peptides

Abstract

The use of efficient and inexpensive substrates (2D matrices) for cultivation and differentiation of nerve cells in vitro is important for the creation of tissue engineering constructs intended for the treatment of nervous system pathologies. Recombinant analogues of the orb-weaver spider dragline-silk proteins spidroins 1 and 2 appear promising in addressing this task. The aim of the study was to evaluate the effect of cell substrates derived from mixtures of recombinant spidroins (RS) rS1/9 and rS2/12 with hybrid proteins (HP) containing rS1/9 monomer fused with biologically active peptides on gene expression levels of key synapse-specific proteins and viability of the human neuroblastoma SH-SY5Y cell line during directed cholinergic differentiation. A two-stage scheme of directed cholinergic differentiation of SH-SY5Y cells using retinoic acid and brain-derived neurotrophic factor (BDNF) was implemented. Cell viability was assessed via MTT assay and crystal violet staining. The mRNA levels of the studied genes were assessed by real-time PCR. Directed differentiation of the SH-SY5Y cells was marked by a significant increase in the gene expression levels of synaptophysin, synapsins I and II, and the postsynaptic protein PSD-95. The highest cell viability and increased PSD-95 expression levels were observed during differentiation on a matrix consisting of RS rS1/9 and rS2/12 mixed with the RGDS peptide (present in extracellular matrix proteins) and heparin-binding peptide (HBP, laminin fragment) containing HPs. The highest efficiency during the differentiation of the SH-SY5Y cells was demonstrated by a matrix consisting of the mixture of RS rS1/9 and rS2/12 and a HP made up by RS rS1/9 monomer fused with RGDS (the ligand of integrins) and HBP (the ligand of growth factors and syndecans). Matrices consisting of RS rS2/12 alone or the mixture of rS2/12 with HP(RGDS) showed lower efficiency, although the use of the GRGGL peptide (which interacts with the neural cell adhesion molecules and is a component of RS rS1/9) led to an increase in efficiency.