The effect of cerebrospinal fluid-derived exosomes on neural differentiation of adipose mesenchymal stem cells in alginate hydrogel scaffold

Cheravi, Mojtaba; Baharara, Javad; Yaghmaei, Parichehreh; Hayati Roudbari, Nasim

doi:10.52547/nbr.8.4.265

The effect of cerebrospinal fluid-derived exosomes on neural differentiation of adipose mesenchymal stem cells in alginate hydrogel scaffold

Authors

Mojtaba Cheravi ¹

Javad Baharara ²

Parichehreh Yaghmaei ¹

Nasim Hayati Roudbari ¹

¹ Department of Biology, faculty of science, Science and Research Branch, Islamic Azad University, Tehran, Iran

² Department of Biology and Research Center for Animal Development Applied Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

https://doi.org/10.52547/nbr.8.4.265

Abstract

Nowadays, researchers have made extensive efforts to find new treatments for nerve damage. Meanwhile, the role of exosomes in cell-cell communication is considered to be a new mechanism. Exosomes can act as suitable differentiating agents. The aim of this study was to investigate the differentiating effect of cerebrospinal fluid-derived exosomes on adipose mesenchymal stem cells in alginate hydrogel. Exosomes were extracted from the cerebrospinal fluid by ultracentrifugation and were then identified by atomic force microscopy (AFM), SEM and DLS technique. In addition, Adipose Mesenchymal Stem cells in alginate hydrogel were treated with different concentrations of exosomes. Cell survival was assessed by MTT and Acridine Orange/Ethidium Bromide methods. Cell differentiation was processed by immunocytochemistry and Real-Time PCR. Examinations confirmed the presence of exosomes with an approximate size of 70 nm. Cell survival results indicate that he ability of cells to survive and proliferate during 14 days. Also, the expression of MAP2 proteins (microtubule-associated protein 2) and Nestin (intermediate filament protein) was confirmed by immunocytochemistry. The results of Real Time - PCR showed that during the seventh and fourteenth days the expression level of MAP2 gene increased and the expression of Nestin gene showed a significant decrease compared to the control group. This study showed that exosomes extracted from cerebrospinal fluid can cause neuronal differentiation of Adipose mesenchymal stem cells in alginate hydrogel scaffolds.

Keywords

extracellular vesicles

hydrogel

immunocytochemistry

tissue engineering

ultracentrifugation

20.1001.1.24236330.1400.8.4.4.9

Bernal, A. & Arranz, L. 2018. Nestin-expressing progenitor cells: function, identity and therapeutic implications. Cellular and Molecular Life Sciences 75: 2177-2195.
Billiet, T., Gevaert, E., De Schryver, T., Cornelissen, M. & Dubruel, P. 2014. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials 35: 49-62.
Bozza, A., Coates, E.E., Incitti, T., Ferlin, K.M., Messina, A., Menna, E., Bozzi, Y., Fisher, J.P. & Casarosa, S. 2014. Neural differentiation of pluripotent cells in 3D alginate-based cultures. Biomaterials 35: 4636-4645.
Capeling, M.M., Czerwinski, M., Huang, S., Tsai, Y.H., Wu, A., Nagy, M.S., Juliar, B., Sundaram, N., Song, Y., Han, W.M., Takayama, S., Alsberg, E., Garcia, A.J., Helmrath, M., Putnam, A.J. & Spence, J.R. 2019. Nonadhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids. Stem Cell Reports 12: 381-394.
Cavo, M., Fato, M., Penuela, L., Beltrame, F., Raiteri, R. & Scaglione, S. 2016. Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model. Scientific Reports 6: 1-13.
Cui, Y., Xiao, Z., Han, J., Sun, J., Ding, W., Zhao, Y., Chen, B., Li, X. & Dai, J. 2012. MiR-125b orchestrates cell proliferation, differentiation and migration in neural stem/progenitor cells by targeting nestin. BMC Neuroscience 116: 1-13.
Farinazzo, A., Turano, E., Marconi, S., Bistaffa, E., Bazzoli, E. & Bonetti, B. 2015. Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches. Cytotherapy 17: 571-578.
Farivar, S., Mohamadzade, Z., Shiari, R. & Fahimzad, A. 2015. Neural differentiation of human umbilical cord mesenchymal stem cells by cerebrospinal fluid. Iranian Journal of Child Neurology 9: 87-93.
Forcales, S.V. 2015. Potential of adipose-derived stem cells in muscular regenerative therapies. Frontiers in Aging Neuroscience 7: 1-12.
Giorgi Silveira, R., Perello Ferrua, C., do Amaral, C.C., Fernandez Garcia, T., de Souza, K.B. & Nedel, F. 2020. MicroRNAs expressed in neuronal differentiation and their associated pathways: Systematic review and bioinformatics analysis. Brain Research Bulletin 157: 140-148.
Gu, X., Ding, F. & Williams, DF. 2014. Neural tissue engineering options for peripheral nerve regeneration. Biomaterials 35: 6143-6156.
Guha, D., Lorenz, D.R., Misra, V., Chettimada, S., Morgello, S. & Gabuzda, D. 2019. Proteomic analysis of cerebrospinal fluid extracellular vesicles reveals synaptic injury, inflammation, and stress response markers in HIV patients with cognitive impairment. Journal of Neuroinflammation 16: 1-19.
Hamzah, R.N., Alghazali, K.M., Biris, A.S. & Griffin, R.J. 2021. Exosome traceability and cell source dependence on composition and Cell-Cell Cross Talk. International Journal of Molecular Sciences 22: 1-17 (5346).
Jin, Q., Wu, P., Zhou, X., Qian, H. & Xu, W. 2021. Extracellular vesicles: novel roles in neurological disorders. Stem Cells International 2021: 1-16.
Khosravizadeh, Z., Razavi, S., Bahramian, H. & Kazemi, M. 2014. The beneficial effect of encapsulated human adipose-derived stem cells in alginate hydrogel on neural differentiation. Journal of Biomedical Materials Research - Part B Applied Biomaterials 102: 749-755.
Lin, H.R, Heish, C.W, Liu, C.H, Muduli, S., Li, H.F., Higuchi, A., Kumar, S.S., Alarfaj, A.A., Munusamy, M.A., Hsu, S.T., Chen, D.C., Benelli, G., Murugan, K., Cheng, N.C., Wang, H.C. & Wu, G.J. 2017. Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods. Scientific Reports 7: 1-13.
Lizarraga-Valderrama, L.R. & Sheridan, G.K. 2021. Extracellular vesicles and intercellular communication in the central nervous system. FEBS Letters 595: 1391-1410.
Luo, L., Hu, D.H, Yin, J.Q. & Xu, R.X. 2018. Molecular mechanisms of transdifferentiation of adipose-derived stem cells into neural cells: Current status and perspectives. Stem Cells International 8: 1-14.
Martins, T.S., Catita, J., Rosa, I.M., Da Cruz e Silva, O.A.B. & Henriques, AG. 2018. Exosome isolation from distinct biofluids using precipitation and column-based approaches. PLoS ONE 13: 1-16.
Mathew, B., Mansuri, M.S., Williams, K.R. & Nairn, AC. 2021. Exosomes as emerging biomarker tools in neurodegenerative and neuropsychiatric disorders—a proteomics perspective. Brain Sciences 11: 1-18.
Rajmohan, D. & Bellmer, D. 2019. Characterization of spirulina-alginate beads formed using ionic gelation. International Journal of Food Science 219: 1-7.
Razavi, S., Jahromi, M., Amirpour, N. & Khosravizadeh, Z. 2014. Effect of sertraline on proliferation and neurogenic differentiation of human adipose-derived stem cells. Advanced Biomedical Research 3: 97-102.
Razavi, S., Khosravizadeh, Z., Bahramian, H. & Kazemi, M. 2015. Time-dependent effect of encapsulating alginate hydrogel on neurogenic potential. Cell Journal 17: 304-311.
Scioli, M.G., Bielli, A., Gentile, P., Mazzaglia, D., Cervelli, V. & Orlandi, A. 2014. The biomolecular basis of adipogenic differentiation of adipose-derived stem cells. International Journal of Molecular Sciences 15: 6517-6526.
Stuendl, A., Kunadt, M., Kruse, N., Bartels, C., Moebius, W., Danzer, K.M., Mollenhauer, B. & Schneider, A. 2016. Induction of α-synuclein aggregate formation by CSF exosomes from patients with parkinson’s disease and dementia with Lewy bodies. Brain 139: 481-494.
Takeda, Y.S. & Xu, Q. 2015. Neuronal differentiation of human mesenchymal stem cells using exosomes derived from differentiating neuronal cells. PLoS ONE 10: 1-15
Upadhyay, R.K. 2017. Role of biological scaffolds, hydro gels and stem cells in tissue regeneration therapy. Advances in Tissue Engineering & Regenerative Medicine 2: 1-15.
Waller, R., Wyles, M., Heath, P.R., Kazoka, M., Wollff, H., Shaw, P.J. & Kirby, J. 2018. Small RNA sequencing of sporadic amyotrophic lateral sclerosis cerebrospinal fluid reveals differentially expressed miRNAs related to neural and glial activity. Frontiers in Neuroscience 11: 1-13.
Yagi, Y., Ohkubo, T., Kawaji, H., Machida, A., Miyata, H., Goda, S., Roy, S., Hayashizaki, Y., Suzuki, H. & Yokota, T. 2017. Next-generation sequencing-based small RNA profiling of cerebrospinal fluid exosomes. Neuroscience Letters 636: 48-57.
Yang, X., Lu, Z., Wu, H., Li, W., Zheng, L. & Zhao, J. 2018. Collagen-alginate as bioink for three-dimensional (3D) cell printing based cartilage tissue engineering. Materials Science and Engineering C 83: 195-201.
Yuan, P., Ding, L., Chen, H., Wang, Y., Li, C., Zhao, S., Yang, X., Ma, Y., Zhu, J., Qi, X., Zhang, Y., Xia, X. & Zheng, J.C. 2021. Neural stem cell-derived exosomes regulate neural stem cell differentiation through miR-9-hes1 axis. Frontiers in Cell and Developmental Biology 9: 1-17.
Zhou, J., Ghoroghi, S., Benito-martin, A., Wu, H., Unachukwu, U.J., Einbond, L.S., Guariglia, S., Peinado, H. & Redenti, S. 2016. Characterization of induced pluripotent stem cell microvesicle genesis, morphology and pluripotent content. Nature Publishing Group 6: 1-10.
Zlotogorski, A., Dan, H., Gavriel, D. & Tuula, C. 2015. Morphological and molecular features of oral fluid ‑ derived exosomes : oral cancer patients versus healthy individuals. Journal of Cancer Research and Clinical Oncology 5: 1-10.