Directed neuronal differentiation of SH-SY5Y human neuroblastoma cells on 2D matrixes containing recombinant spidroins modified with cell adhesion peptides

Kurko O.D.

National Research Center «Kurchatov Institute»

ORCID: 0009-0005-5206-0587

Davydova L.I.

National Research Center «Kurchatov Institute»”

Sidoruk K.V.

National Research Center «Kurchatov Institute»”

ORCID: 0000-0001-8952-5409

Grivennikov I.A.

National Research Center «Kurchatov Institute»

ORCID: 0000-0003-0128-5661

Debabov V.G.

National Research Center «Kurchatov Institute»”

ORCID: 0000-0001-7577-7437

Bogush V.G.

National Research Center «Kurchatov Institute»”;
All-Russia Research Institute of Agricultural Biotechnology

ORCID: 0000-0002-5969-3855

Tarantul V.Z.

National Research Center «Kurchatov Institute»

ORCID: 0000-0002-5525-0395

Dolotov O.V.

National Research Center «Kurchatov Institute»;
Lomonosov Moscow State University

ORCID: 0000-0003-3187-6331

Directed neuronal differentiation of SH-SY5Y human neuroblastoma cells on 2D matrixes containing recombinant spidroins modified with cell adhesion peptides

Authors:

Kurko O.D., Davydova L.I., Sidoruk K.V., Grivennikov I.A., Debabov V.G., Bogush V.G., Tarantul V.Z., Dolotov O.V.

More about the authors

Journal: Molecular Genetics, Microbiology and Virology. 2023;41(3): 16‑24

DOI: 10.17116/molgen20234103116

Downloaded: 14

Download PDF (RU)

Contact the author

Table of contents

To cite this article:

Kurko OD, Davydova LI, Sidoruk KV, et al. . Directed neuronal differentiation of SH-SY5Y human neuroblastoma cells on 2D matrixes containing recombinant spidroins modified with cell adhesion peptides. Molecular Genetics, Microbiology and Virology. 2023;41(3):16‑24. (In Russ.)
https://doi.org/10.17116/molgen20234103116

Подписка 2025-2 (Molecular Genetics, Microbiology and Virology)

Close metadata

AIM

The use of efficient and inexpensive coatings (2D matrices) for the cultivation and differentiation of nerve cells in vitro is important for the development of tissue-engineered constructs intended for the treatment of pathologies of the nervous system. Promising for such constructs are recombinant analogs of the dragline silk fiber of the orb-weaving spiders web, namely, spidroins 1 and 2. The aim of the study was to evaluate the effects of coatings obtained from mixtures of recombinant spidroins (RSs) rS1/9 and rS2/12 with hybrid proteins (HPs) containing the rS1/9 monomer fused with biologically active peptides on the levels of expression of key synapse-specific genes and viability of SH-SY5Y human neuroblastoma cells in directed cholinergic differentiation.

MATERIAL AND METHODS

A two-stage scheme of directed cholinergic differentiation using retinoic acid and brain-derived neurotrophic factor (BDNF) was applied. Cell viability was assessed using the MTT test and crystal violet staining. The expression levels of the studied genes were assessed using real-time PCR.

RESULTS

Directed differentiation of SH-SY5Y cells was accompanied by a significant increase in the expression levels of the genes of synaptophysin, synapsins I, II, and the postsynaptic protein PSD-95. The highest cell viability and increased levels of PSD-95 expression were found during differentiation on a coating consisting of a mixture of RSs rS1/9 and rS2/12 with HPs containing the RGDS peptide (present in extracellular matrix proteins) and the heparin-binding peptide HBP (a fragment of laminin).

CONCLUSION

The coating consisting of a mixture of RSs rS1/9 and rS2/12 and HP based on the RS monomer rS1/9 fused with RGDS (a ligand for integrins) and HBP (a ligand for growth factors and syndecans) showed the highest efficiency in directed neuronal differentiation of SH-SY5Y cells. Coatings consisting of rS2/12 alone or a mixture of rS2/12 with HP(RGDS) demonstrated less efficiency, but the fusion with the GRGGL peptide (which interacts with NCAMs and is a fragment of rS1/9) resulted in an increase in efficiency.

Keywords:

combined matrix

recombinant spidroins

biologically active peptides

human neuroblastoma

directed neuronal differentiation

synapse-specific proteins

viability of neurons

Authors:

Kurko O.D.

National Research Center «Kurchatov Institute»

ORCID: 0009-0005-5206-0587

Davydova L.I.

National Research Center «Kurchatov Institute»”

Sidoruk K.V.

National Research Center «Kurchatov Institute»”

ORCID: 0000-0001-8952-5409

Grivennikov I.A.

National Research Center «Kurchatov Institute»

ORCID: 0000-0003-0128-5661

Debabov V.G.

National Research Center «Kurchatov Institute»”

ORCID: 0000-0001-7577-7437

Bogush V.G.

National Research Center «Kurchatov Institute»”;
All-Russia Research Institute of Agricultural Biotechnology

ORCID: 0000-0002-5969-3855

Tarantul V.Z.

National Research Center «Kurchatov Institute»

ORCID: 0000-0002-5525-0395

Dolotov O.V.

National Research Center «Kurchatov Institute»;
Lomonosov Moscow State University

ORCID: 0000-0003-3187-6331

Received:

06.04.2023

Accepted:

10.07.2023

List of references:

Gholipourmalekabadi M, Zhao S, Harrison BS, Mozafari M, Seifalian AM. Oxygen-Generating Biomaterials: A New, Viable Paradigm for Tissue Engineering? Trends Biotechnol. 2016;34(12):1010-1021. https://doi.org/10.1016/j.tibtech.2016.05.012
Blackledge TA. Spider silk: a brief review and prospectus on research linking biomechanics and ecology in draglines and orb webs. J Arachnology. 2012;40(1):1-12, 12.
Debabov VG, Bogush VG. Recombinant Spidroins as the Basis for New Materials. ACS Biomater Sci Eng. 2020;6(7):3745-3761. https://doi.org/10.1021/acsbiomaterials.0c00109
Rising A, Widhe M, Johansson J, Hedhammar M. Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications. Cell Mol Life Sci. 2011;68(2):169-184. https://doi.org/10.1007/s00018-010-0462-z
Bogush VG, Sokolova OS, Davydova LI, Klinov DV, Sidoruk KV, Esipova NG, et al. A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology. 2009;4(1):17-27. https://doi.org/10.1007/s11481-008-9129-z
Jansson R, Thatikonda N, Lindberg D, Rising A, Johansson J, Nygren P, Hedhammar M. Recombinant spider silk genetically functionalized with affinity domains. Biomacromolecules. 2014;15(5):1696-706. https://doi.org/10.1021/bm500114e
Matsuda A, Kobayashi H, Itoh S, Kataoka K, Tanaka J. Immobilization of laminin peptide in molecularly aligned chitosan by covalent bonding. Biomaterials. 2005;26(15):2273-2279. https://doi.org/10.1016/j.biomaterials.2004.07.032
Mauri E, Sacchetti A, Vicario N, Peruzzotti-Jametti L, Rossi F, Pluchino S. Evaluation of RGD functionalization in hybrid hydrogels as 3D neural stem cell culture systems. Biomaterials science. 2018;6(3):501-510. https://doi.org/10.1039/c7bm01056g
Moisenovich MM, Silachev DN, Moysenovich AM, Arkhipova AY, Shaitan KV, Bogush VG, et al. Effects of Recombinant Spidroin rS1/9 on Brain Neural Progenitors After Photothrombosis-Induced Ischemia. Frontiers in cell and developmental biology. 2020;8:823. https://doi.org/10.3389/fcell.2020.00823
Hirano Y, Okuno M, Hayashi T, Goto K, Nakajima A. Cell-attachment activities of surface immobilized oligopeptides RGD, RGDS, RGDV, RGDT, and YIGSR toward five cell lines. Journal of biomaterials science Polymer edition. 1993;4(3):235-243. https://doi.org/10.1163/156856293x00546
Wohlrab S, Müller S, Schmidt A, Neubauer S, Kessler H, Leal-Egaña A, Scheibel T. Cell adhesion and proliferation on RGD-modified recombinant spider silk proteins. Biomaterials. 2012;33(28):6650-6659. https://doi.org/10.1016/j.biomaterials.2012.05.069
Timpl R, Rohde H, Robey PG, Rennard SI, Foidart JM, Martin GR. Laminin--a glycoprotein from basement membranes. The Journal of biological chemistry. 1979;254(19):9933-9937.
Ishihara J, Ishihara A, Fukunaga K, Sasaki K, White MJV, Briquez PS, Hubbell JA. Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing. Nature communications. 2018;9(1):2163. https://doi.org/10.1038/s41467-018-04525-w
An B, Tang-Schomer M, Huang W, He J, Jones J, Lewis RV, Kaplan DL. Physical and biological regulation of neuron regenerative growth and network formation on recombinant dragline silks. Biomaterials. 2015;48:137-146. https://doi.org/10.1016/j.biomaterials.2015.01.044
Goldie BJ, Barnett MM, Cairns MJ. BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation. Frontiers in cellular neuroscience. 2014;8325.
de Medeiros LM, De Bastiani MA, Rico EP, Schonhofen P, Pfaffenseller B, Wollenhaupt-Aguiar B,et al. Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer’s Disease Studies. Molecular neurobiology. 2019;56(11):7355-7367. https://doi.org/10.1007/s12035-019-1605-3
Gimenez-Cassina A, Lim F, Diaz-Nido J. Differentiation of a human neuroblastoma into neuron-like cells increases their susceptibility to transduction by herpesviral vectors. Journal of neuroscience research. 2006;84(4):755-767. https://doi.org/10.1002/jnr.20976
Revkova VA, Sidoruk KV, Kalsin VA, Melnikov PA, Konoplyannikov MA, Kotova S, et al. Spidroin Silk Fibers with Bioactive Motifs of Extracellular Proteins for Neural Tissue Engineering. ACS omega. 2021;6(23):15264-15273. https://doi.org/10.1021/acsomega.1c01576
Novosadova EV, Dolotov OV, Novosadova LV, Davydova LI, Sidoruk KV, Arsenyeva EL, et al. Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells. International journal of molecular sciences. 2023;24(5)4871. https://doi.org/10.3390/ijms24054871
Lerman MJ, Lembong J, Muramoto S, Gillen G, Fisher JP. The Evolution of Polystyrene as a Cell Culture Material. Tissue Engineering Part B: Reviews. 2018;24(5):359-372. https://doi.org/10.1089/ten.teb.2018.0056
Feoktistova M, Geserick P, Leverkus M. Crystal Violet Assay for Determining Viability of Cultured Cells. Cold Spring Harbor protocols. 2016;2016(4):pdb prot087379. https://doi.org/10.1101/pdb.prot087379
Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, Boyd MR. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer research. 1991;51(10):2515-2520.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif). 2001;25(4):402-408. https://doi.org/10.1006/meth.2001.1262
Goudarzi F, Tayebinia H, Karimi J, Habibitabar E, Khodadadi I. Calcium: A novel and efficient inducer of differentiation of adipose-derived stem cells into neuron-like cells. Journal of cellular physiology. 2018;233(11):8940-8951. https://doi.org/10.1002/jcp.26826
Imai C, Sugai T, Iritani S, Niizato K, Nakamura R, Makifuchi T, et al. A quantitative study on the expression of synapsin II and N-ethylmaleimide-sensitive fusion protein in schizophrenic patients. Neuroscience letters. 2001;305(3):185-188. https://doi.org/10.1016/s0304-3940(01)01844-4
Sen A, Hongpaisan J, Wang D, Nelson TJ, Alkon DL. Protein Kinase C (PKC) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95. The Journal of biological chemistry. 2016;291(32):16462-16476. https://doi.org/10.1074/jbc.M116.730440
Artyukhov AS, Dashinimaev EB, Tsvetkov VO, Bolshakov AP, Konovalova EV, Kolbaev SN, et al. New genes for accurate normalization of qRT-PCR results in study of iPS and iPS-derived cells. Gene. 2017;626:234-240. https://doi.org/10.1016/j.gene.2017.05.045
Song Y, Zuo Y. Occurrence of HHIP gene CpG island methylation in gastric cancer. Oncology letters. 2014;8(5):2340-2344. https://doi.org/10.3892/ol.2014.2518
Tojima T, Ito E. Signal transduction cascades underlying de novo protein synthesis required for neuronal morphogenesis in differentiating neurons. Progress in neurobiology. 2004;72(3):183-193. https://doi.org/10.1016/j.pneurobio.2004.03.002
Son G, Han J. Roles of mitochondria in neuronal development. BMB reports. 2018;51(11):549-556. https://doi.org/10.5483/BMBRep.2018.51.11.226

Close metadata