Transdifferentiation of rat astrocytes into neurons induced by ectodermal mesenchymal stem cells-derived extracellular vesicles

doi:10.12307/2022.764

Abstract

Abstract: BACKGROUND: Spinal cord injury will lead to the proliferation of astrocytes, resulting in glial scar that hinders the formation of axons, and the local inflammatory microenvironment further aggravates the death of neurons. Therefore, converting astrocytes into neurons reduces the proliferation of astrocytes and increases the number of neurons — killing two birds with one stone. However, it has not been reported whether ectodermal mesenchymal stem cells-derived extracellular vesicles can induce astrocytes to differentiate into neurons.
OBJECTIVE: To explore whether ectodermal mesenchymal stem cells-derived extracellular vesicles can induce astrocytes to transdifferentiate into neurons.
METHODS: (1) Ectodermal mesenchymal stem cells derived from nasal mucosa tissue of SD rats were cultured by the tissue explant culture and identified according to their morphology and immunofluorescence. The cell supernatant was collected to extract extracellular vesicles, which were identified by particle size analysis, transmission electron microscopy, and western blot assay. (2) The astrocytes of SD rats were cultured and identified by trypsin digestion technique. (3) Astrocytes were divided into two groups for intervention. Ectodermal mesenchymal stem cells-derived extracellular vesicles (mass concentration:
1 g/L, volume: 66 μL added into 2 mL medium) and PBS of the same volume were added into astrocytes for 72 hours. (4) The expression levels of neuron markers neuron-specific enolase and neurofilament 200 were detected by immunofluorescence, real-time fluorescence quantitative PCR, and western blot assay.
RESULTS AND CONCLUSION: (1) Ectodermal mesenchymal stem cells presented typical spindle shape and high expression of marker proteins CD44, Nestin, and Vimentin. The ectodermal mesenchymal stem cells-derived extracellular vesicles exhibited classic tea receptor-like morphology. The particle size distribution ranged from 30 nm to 200 nm. Western blot assay demonstrated positive expression of marker proteins CD9, CD63, and TSG101. (2) Astrocytes were star-shaped and highly expressed the marker protein glial fibrillary acidic protein. (3) Compared with the control group, there were more neuron-like cells in the ectodermal mesenchymal stem cells-derived extracellular vesicles group. The cell bodies were spindle shaped and full; the processes increased and became longer; and the neuron-specific enolase and neurofilament 200 were highly expressed (P < 0.05). (4) The above results suggest that ectodermal mesenchymal stem cells-derived extracellular vesicles induce astrocytes to transdifferentiate into neurons.

Key words: mesenchymal stem cell, extracellular vesicle, central nervous system, astrocytes, neurons, neuron-specific enolase, neurofilament 200, transdifferentiation

CLC Number:

Guan Shihao, Huang Yonghui, Gong Aihua, Cao Xingbing, Sun Haitao, Cai Chuang. Transdifferentiation of rat astrocytes into neurons induced by ectodermal mesenchymal stem cells-derived extracellular vesicles[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(30): 4840-4846.

Figures/Tables 6

References

[1] HOU B, ZHANG Y, LIANG P, et al. Inhibition of the NLRP3-inflammasome prevents cognitive deficits in experimental autoimmune encephalomyelitis mice via the alteration of astrocyte phenotype. Cell Death Dis. 2020;11(5):377.
[2] MURRAY CE, GAMI-PATEL P, GKANATSIOU E, et al. The presubiculum is preserved from neurodegenerative changes in Alzheimer’s disease. Acta Neuropathol Commun. 2018;6(1):62.
[3] CHEN J, HU R, GE H, et al. G-protein-coupled receptor 30-mediated antiapoptotic effect of estrogen on spinal motor neurons following injury and its underlying mechanisms. Mol Med Rep. 2015;12(2):1733-1740.
[4] SARKIS GA, MANGAONKAR MD, MOGHIEB A, et al. The Application of Proteomics to Traumatic Brain and Spinal Cord Injuries. Curr Neurol Neurosci Rep. 2017;17(3):23.
[5] LEE JS, HSU YH, CHIU YS, et al. Anti-IL-20 antibody improved motor function and reduced glial scar formation after traumatic spinal cord injury in rats. J Neuroinflammation. 2020;17(1):156.
[6] LI P, GAO Y, LI X, et al. mRNA and miRNA expression profile reveals the role of miR-31 overexpression in neural stem cell. Sci Rep. 2020; 10(1):17537.
[7] QU J, ZHANG H. Roles of Mesenchymal Stem Cells in Spinal Cord Injury. Stem Cells Int. 2017;2017:5251313.
[8] TSAI MJ, LIOU DY, LIN YR, et al. Attenuating Spinal Cord Injury by Conditioned Medium from Bone Marrow Mesenchymal Stem Cells. J Clin Med. 2018;8(1):23.
[9] COFANO F, BOIDO M, MONTICELLI M, et al. Mesenchymal Stem Cells for Spinal Cord Injury: Current Options, Limitations, and Future of Cell Therapy. Int J Mol Sci. 2019;20(11):2698.
[10] LIAU LL, LOOI QH, CHIA WC, et al. Treatment of spinal cord injury with mesenchymal stem cells. Cell Biosci. 2020;10:112.
[11] 黄佩琦,卓毅,段答,等.嗅粘膜间充质干细胞与嗅粘膜嗅鞘细胞在人鼻粘膜中分布的研究[J].湖南师范大学学报(医学版),2020, 17(5):10-14.
[12] HUANG H, ZOU X, ZHONG L, et al. CRISPR/dCas9-mediated activation of multiple endogenous target genes directly converts human foreskin fibroblasts into Leydig-like cells. J Cell Mol Med. 2019;23(9):6072-6084.
[13] LIANG B, LIANG JM, DING JN, et al. Dimethyloxaloylglycine-stimulated human bone marrow mesenchymal stem cell-derived exosomes enhance bone regeneration through angiogenesis by targeting the AKT/mTOR pathway. Stem Cell Res Ther. 2019;10(1):335.
[14] DENG Z, WANG Y, ZHOU L, et al. High salt-induced activation and expression of inflammatory cytokines in cultured astrocytes. Cell Cycle. 2017;16(8):785-794.
[15] YANG H, YAN H, LI X, et al. Inhibition of Connexin 43 and Phosphorylated NR2B in Spinal Astrocytes Attenuates Bone Cancer Pain in Mice. Front Cell Neurosci. 2018;12:129.
[16] SHIGETOMI E, SAITO K, SANO F, et al. Aberrant Calcium Signals in Reactive Astrocytes: A Key Process in Neurological Disorders. Int J Mol Sci. 2019;20(4):996.
[17] YU Y, YAN Y, LUO Z, et al. Effects of human umbilical cord blood CD34+ cell transplantation in neonatal hypoxic-ischemia rat model. Brain Dev. 2019;41(2):173-181.
[18] HUANG Y, TAN S. Direct lineage conversion of astrocytes to induced neural stem cells or neurons. Neurosci Bull. 2015;31(3):357-367.
[19] SHIH CH, LACAGNINA M, LEUER-BISCIOTTI K, et al. Astroglial-derived periostin promotes axonal regeneration after spinal cord injury. J Neurosci. 2014;34(7):2438-2443.
[20] BRULET R, MATSUDA T, ZHANG L, et al. NEUROD1 Instructs Neuronal Conversion in Non-Reactive Astrocytes. Stem Cell Reports. 2017;8(6): 1506-1515.
[21] ROBINSON M, FRASER I, MCKEE E, et al. Transdifferentiating Astrocytes Into Neurons Using ASCL1 Functionalized With a Novel Intracellular Protein Delivery Technology. Front Bioeng Biotechnol. 2018;6:173.
[22] 凌华军,王其友,林伟文,等.骨髓间充质干细胞来源外泌体保护软骨细胞延缓骨关节炎的发生发展[J].中国组织工程研究,2021, 25(31):4964-4969.
[23] ZHAO AG, SHAH K, CROMER B, et al. Mesenchymal Stem Cell-Derived Extracellular Vesicles and Their Therapeutic Potential. Stem Cells Int. 2020;2020:8825771.
[24] LIU W, RONG Y, WANG J, et al. Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization. J Neuroinflammation. 2020;17(1):47.
[25] KOMATSU F, FARKAS I, AKATSU H, et al. Potential neural progenitor cells in fetal liver and regenerating liver. Cytotechnology. 2008;56(3):209-217.
[26] NAKANO M, FUJIMIYA M. Potential effects of mesenchymal stem cell derived extracellular vesicles and exosomal miRNAs in neurological disorders. Neural Regen Res. 2021;16(12):2359-2366.
[27] 朱东京,鲜盼盼,王甜,等.间充质干细胞外泌体对海马星形胶质细胞活化的抑制作用研究[J].中华细胞与干细胞杂志(电子版), 2021,11(2):99-105.
[28] RONG Y, LIU W, WANG J, et al. Neural stem cell-derived small extracellular vesicles attenuate apoptosis and neuroinflammation after traumatic spinal cord injury by activating autophagy. Cell Death Dis. 2019;10(5):340.
[29] ROMANELLI P, BIELER L, SCHARLER C, et al. Extracellular Vesicles Can Deliver Anti-inflammatory and Anti-scarring Activities of Mesenchymal Stromal Cells After Spinal Cord Injury. Front Neurol. 2019;10:1225.
[30] LU Y, ZHOU Y, ZHANG R, et al. Bone Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Recovery Following Spinal Cord Injury via Improvement of the Integrity of the Blood-Spinal Cord Barrier. Front Neurosci. 2019;13:209.
[31] LIU FT, XU SM, XIANG ZH, et al. Molecular hydrogen suppresses reactive astrogliosis related to oxidative injury during spinal cord injury in rats. CNS Neurosci Ther. 2014;20(8):778-786.
[32] YU B, YAO C, WANG Y, et al. The Landscape of Gene Expression and Molecular Regulation Following Spinal Cord Hemisection in Rats. Front Mol Neurosci. 2019;12:287.
[33] YOKOYAMA A, SAKAMOTO A, KAMEDA K, et al. NG2 proteoglycan-expressing microglia as multipotent neural progenitors in normal and pathologic brains. Glia. 2006;53(7):754-768.
[34] ZAREI-KHEIRABADI M, HESARAKI M, KIANI S, et al. In vivo conversion of rat astrocytes into neuronal cells through neural stem cells in injured spinal cord with a single zinc-finger transcription factor. Stem Cell Res Ther. 2019;10(1):380.
[35] WANG J, RONG Y, JI C, et al. MicroRNA-421-3p-abundant small extracellular vesicles derived from M2 bone marrow-derived macrophages attenuate apoptosis and promote motor function recovery via inhibition of mTOR in spinal cord injury. J Nanobiotechnology. 2020; 18(1):72.
[36] RUPPERT KA, NGUYEN TT, PRABHAKARA KS, et al. Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modify Microglial Response and Improve Clinical Outcomes in Experimental Spinal Cord Injury. Sci Rep. 2018;8(1):480.
[37] XIAO X, LI W, RONG D, et al. Human umbilical cord mesenchymal stem cells-derived extracellular vesicles facilitate the repair of spinal cord injury via the miR-29b-3p/PTEN/Akt/mTOR axis. Cell Death Discov. 2021;7(1):212.
[38] DING ZB, SONG LJ, WANG Q, et al. Astrocytes: a double-edged sword in neurodegenerative diseases. Neural Regen Res. 2021;16(9):1702-1710.
[39] YIN JC, ZHANG L, MA NX, et al. Chemical Conversion of Human Fetal Astrocytes into Neurons through Modulation of Multiple Signaling Pathways. Stem Cell Reports. 2019;12(3):488-501.
[40] LIU S, LIU D, CHEN C, et al. MSC Transplantation Improves Osteopenia via Epigenetic Regulation of Notch Signaling in Lupus. Cell Metab. 2015; 22(4):606-618.
[41] COLLETTI M, TOMAO L, GALARDI A, et al. Neuroblastoma-secreted exosomes carrying miR-375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells. J Extracell Vesicles. 2020; 9(1):1774144.