The Effect of Culture Dimensionality and Brain Extracellular Matrix in Neuronal Differentiation

Year 2023, , 142 - 153, 21.12.2023

Duygu Turan Sorhun , Ece Öztürk

https://doi.org/10.26650/EurJBiol.2023.1317681

Abstract

Objective: Neuroblastoma cells are frequently used in neuroscience studies due to their human origin and ability of extensive propagation compared to animal-derived primary neuron cultures. Although they are tumor-derived, they exhibit neuronal differentiation capability in the presence of several agents including retinoid acid. Several studies have quested for successful differentiation protocols and faithful representation of neuronal characteristics. However, they predominantly pursued conventional two-dimensional (2D) cultures where the role of three-dimensional (3D) tissue microenvironment and cell-matrix interactions remained unknown. In this study, we investigated the effect of culture dimensionality and native brain extracellular matrix (ECM) on neuronal differentiation of neuroblastoma cells.

Materials and Methods: Decellularized brain ECM hydrogels offer a physiologically relevant in vitro 3D culture platform with the representation of key biochemical and biophysical aspects of the native tissue microenvironment for modeling cellular processes. We cultured SH-SY5Y cells on 2D or as encapsulated in 3D decellularized brain ECM hydrogels and assessed them for morphological shift, neurite extension, and expression of neuronal, synaptic, astrocytic, cholinergic, stemness, proto-oncogene and neuropathological markers.

Results: Our findings demonstrate that the 3D brain ECM microenvironment distinctly affects the differentiation process compared to conventional culturing. In 3D ECM, neuronal differentiation occurred as in 2D, with upregulation of neuronal markers, changein cell morphology, and promotion of neurite extension. However, during differentiation, maintenance of stemness was observed in a 3D-specific manner. Furthermore, 3D differentiation promoted significant upregulation of astrocytic and synaptic markers which was not observed in 2D.

Conclusion: This study highlights the importance of physio-mimetic 3D brain models.

Keywords

Neuronal differentiation, extracellular matrix, brain tissue engineering, decellularization, hydrogels

References

• Karamanos NK, Theocharis AD, Piperigkou Z, et al. A guide to the composition and functions of the extracellular matrix. FEBS J. 2021;288(24):6850-6912. doi:10.1111/febs.15776 google scholar
• Kim BS, Das S, Jang J, Cho D-W. Decellularized Extracellular Matrix-based bioinks for engineering tissue- and organ-specific microenvironments. Chem Rev. 2020;120(19):10608-10661. google scholar
• Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as abi-ological scaffold material: Structure and function. Acta Biomater. 2009;5(1):1-13. google scholar
• Freytes DO, Martin J, Velankar SS, Lee AS, Badylak SF. Prepara-tion and rheological characterization of a gel form of the porcine urinary bladder matrix. Biomaterials. 2008;29(11):1630-1637. google scholar
• DeQuach JA, Yuan SH, Goldstein LS, Christman KL. Decellular-ized porcine brain matrix for cell culture and tissue engineering scaffolds. Tissue Eng Part A. 2011;17(21-22):2583-2592. google scholar
• Jin Y, Lee JS, Kim J, et al. Three-dimensional brain-like microen-vironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons. Nat Biomed Eng. 2018;2(7):522-539. google scholar
• Cembran A, Bruggeman KF, Williams RJ, Parish CL, Nisbet DR. Biomimetic materials and their utility in modeling the 3-dimensional neural environment. iScience. 2020;23(1):100788. doi:10.1016/j.isci.2019.100788 google scholar
• Sood D, Cairns DM, Dabbi JM, et al. Functional maturation of human neural stem cells in a 3D bioengineered brain model en-riched with fetal brain-derived matrix. Sci Rep. 2019;9(1):17874. doi:10.1038/s41598-019-54248-1 google scholar
• Hebisch M, Klostermeier S, Wolf K, et al. The impact of the cellular environment and aging on modeling Alzheimer’s dis-ease in 3D cell culture models. Adv Sci. 2023;10(8):2205037. doi:10.1002/advs.202205037 google scholar
• Jain D, Mattiassi S, Goh EL, Yim EKF. Extracellular matrix and biomimetic engineering microenvironment for neuronal differen-tiation. Neural Regen Res. 2020;15(4):573-585. google scholar
• Nicolas J, Magli S, Rabbachin L, Sampaolesi S, Nicotra F, Russo L. 3D Extracellular matrix mimics: Fundamental concepts and role of materials chemistry to influence stem cell fate. Biomacro-molecules. 2020;21(6):1968-1994. google scholar
• Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS. Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 1978;38(11Part1):3751-3757. google scholar
• Kovalevich J, Langford D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol Biol. 2013;1078:9-21. google scholar
• Barth M, Toto Nienguesso A, Navarrete Santos A, Schmidt C. Quantitative proteomics and in-cell cross-linking reveal cellu-lar reorganisation during early neuronal differentiation of SH-SY5Y cells. Commun Biol. 2022;5(1):551. doi:10.1038/s42003-022-03478-7 google scholar
• de Medeiros LM, De Bastiani MA, Rico EP, 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. Mol Neurobiol. 2019;56(11):7355-7367. google scholar
• Shipley MM, Mangold CA, Szpara ML. Differentiation of the SH-SY5Y human neuroblastoma cell line. J Vis Exp. 2016;(108):e53193. doi:10.3791/53193 google scholar
• Qiao J, Paul P, Lee S, et al. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation. Biochem Biophy Res Commun. 2012;424(3):421-426. google scholar
• Bottenstein JE, Sato GH. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci USA. 1979;76(1):514-517. google scholar
• Sünwoldt J, Bosche B, Meisel A, Mergenthaler P. Neu-ronal cultu re microenvironments determine preferences in bioenergetic pathway use. Front Mol Neurosci. 2017;10:305. doi:10.3389/fnmol.2017.00305 google scholar
• Xie H-r, Hu L-s, Li G-y. SH-SY5Y human neuroblastoma cell line: In vitro cell model of dopaminergic neurons in Parkinson’s disease. Chin Med J. 2010;123(08):1086-1092. google scholar
• Dehmelt L, Halpain S. The MAP2/Tau family of microtubule-associated proteins. Genome Biol. 2004;6(1):204. doi:10.1186/gb-2004-6-1-204 google scholar
• Kaech S, Ludin B, Matus A. Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins. Neuron. 1996;17(6):1189-1199. google scholar
• Katsetos CD, Herman MM, Mörk SJ. Class III^-tubulin in human development and cancer. Cell motil Cytoskelet. 2003;55(2):77-96. google scholar
• Gusel’nikova VV, Korzhevskiy DE. NeuN As a neuronal nu-clear antigen and neuron differentiation marker. Acta Nat. 2015;7(2):42-47. google scholar
• Cassiman D, van Pelt J, De Vos R, et al. Synaptophysin: A novel marker for human and rat hepatic stellate cells. Am J Pathol. 1999;155(6):1831-1839. google scholar
• White DN, Stowell MHB. Room for two: The synap-tophysin/synaptobrevin complex. Front Synaptic Neurosci. 2021;13:740318. doi:10.3389/fnsyn.2021.740318. google scholar
• Graham V, Khudyakov J, Ellis P, Pevny L. SOX2 functions to maintain neural progenitor identity. Neuron. 2003;39(5):749-765. google scholar
• Bunone G, Borrello MG, Picetti R, et al. Induction of RET proto-oncogene expression in neuroblastoma cells precedes neuronal differentiation and is not mediated by protein synthesis. Exp Cell Res. 1995;217(1):92-99. google scholar
• Tahira T, Ishizaka Y, Itoh F, Nakayasu M, Sugimura T, Nagao M. Expression of the ret proto-oncogene in human neuroblastoma cell lines and its increase during neuronal differentiation induced by retinoic acid. Oncogene. 1991;6(12):2333-2338. google scholar
• Oda Y. Choline acetyltransferase: The structure, distribution and pathologic changes in the central nervous system. Pathol Int. 1999;49(11):921-937. google scholar
• Yang Z, Wang KK. Glial fibrillary acidic protein: From inter-mediate filament assembly and gliosis to neurobiomarker. Trends Neurosci. 2015;38(6):364-374. google scholar
• Müller UC, Deller T, Korte M. Not just amyloid: Physiologi-cal functions of the amyloid precursor protein family. Nat Rev Neurosci. 2017;18(5):281-298. doi:10.1038/nrn.2017.29 google scholar
• Bagaria J, Bagyinszky E, An SSA. Genetics, functions, and clinical impact of presenilin-1 (psen1) gene. Int J Mol Sci. 2022;23(18):10970. doi:10.3390/ijms231810970 google scholar
• Bayeva N, Coll E, Piskareva O. Differentiating neuroblas-toma: A systematic review of the retinoic acid, its deriva-tives, and synergistic interactions. J Pers Med. 2021;11(3): 211. doi:10.3390/jpm11030211 google scholar
• Centeno EGZ, Cimarosti H, Bithell A. 2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegen-erative disease modelling. Mol Neurodegener. 2018;13(1):27. doi:10.1186/s13024-018-0258-4 google scholar
• Lovett ML, Nieland TJ, Dingle YTL, Kaplan DL. In-novations in 3D tissue models of human brain physiol-ogy and diseases. Adv Funct Mater. 2020;30(44):1909146. doi:10.1002/adfm.201909146 google scholar
• Roth JG, Huang MS, Li TL, et al. Advancing models of neural de-velopment with biomaterials. Nat Rev Neurosci. 2021;22(10):593-615. doi:10.1038/s41583-021-00496-y google scholar
• Cai A, Lin Z, Liu N, et al. Neuroblastoma SH-SY5Y cell differ-entiation to mature neuron by AM580 treatment. Neurochem Res. 2022;47(12):3723-3732. google scholar
• Bell N, Hann V, Redfern CPF, Cheek TR. Store-operated Ca2+ entry in proliferating and retinoic acid-differentiated N- and S-type neuroblastoma cells. Biochim Biophys Acta Mol Cell Res. 2013;1833(3):643-651. google scholar
• Dhara SK, Stice SL. Neural differentiation of human embryonic stem cells. J Cell Biochem. 2008;105(3):633-640. google scholar
• Lasher RS, Zagon IS. The effect of potassium on neuronal differ-entiation in cultures of dissociated newborn rat cerebellum. Brain Research. 1972;41(2):482-488. google scholar
• Yang T, Uhler M. KCl-induced depolarization facilitates neuronal differentiation of P19 embryonic carcinoma cells. UMURF. 2013; (6): 52-58. google scholar
• Przyborski SA, Cambray-Deakin MA. Developmental regulation of MAP2 variants during neuronal differentiation in vitro. Brain Res Dev Brain Res. 1995;89(2):187-201. google scholar
• Forster JI, Köglsberger S, Trefois C, et al. Characterization of differentiated SH-SY5Y as neuronal screening model reveals in-creased oxidative vulnerability. SLAS Discovery. 2016;21(5):496-509. google scholar
• Craig BT, Rellinger EJ, Alvarez AL, Dusek HL, Qiao J, Chung DH. Induced differentiation inhibits sphere formation in neurob-lastoma. Biochem Biophys Res Commun. 2016;477(2):255-259. google scholar
• Long KR, Huttner WB. How the extracellular matrix shapes neu-ral development. Royal Society Open Biol. 2019;9(1):180216. doi:10.1098/rsob.180216 google scholar
• Ransanz LC, Van Altena PF, Heine VM, Accardo A. Engineered cell culture microenvironments for mechanobiology studies of brain neural cells. Front Bioeng Biotechnol. 2022;10:1096054. doi:10.3389/fbioe.2022.1096054 google scholar
• Hu Y, Huang G, Tian J, et al. Matrix stiffness changes affect astrocyte phenotype in an in vitro injury model. NPG Asia Mater. 2021;13(1):35. doi:10.1038/s41427-021-00304-0 google scholar
• Saraswathibhatla A, Indana D, Chaudhuri O. Cell-extracellular matrix mechanotransduction in 3D. Nat Rev Mol Cell Biol. 2023;24(7):495-516. google scholar
• Kim HN, Choi N. Consideration of the mechanical properties of hydrogels for brain tissue engineering and brain-on-a-chip. BioChip J. 2019;13(1):8-19. google scholar
• Madl CM, LeSavage BL, Dewi RE, et al. Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remod-elling. Nature Materials. 2017;16(12):1233-1242. google scholar
• Chaudhuri O, Gu L, Klumpers D, et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat Mater. 2016;15(3):326-334. google scholar
• Khetan S, Guvendiren M, Legant WR, Cohen DM, Chen CS, Burdick JA. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nature Mater. 2013;12(5):458-465. google scholar
• Ma Y, Han T, Yang Q, et al. Viscoelastic cell microenvi-ronment: Hydrogel-based strategy for recapitulating dynamic ECM mechanics. Adv Funct Mater. 2021;31(24):2100848. doi:10.1002/adfm.202100848 google scholar