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Single-cell RNA sequencing reveals Nestin+ active neural stem cells outside the central canal after spinal cord injury

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Abstract

Neural stem cells (NSCs) in the spinal cord hold great potential for repair after spinal cord injury (SCI). The ependyma in the central canal (CC) region has been considered as the NSCs source in the spinal cord. However, the ependyma function as NSCs after SCI is still under debate. We used Nestin as a marker to isolate potential NSCs and their immediate progeny, and characterized the cells before and after SCI by single-cell RNA-sequencing (scRNA-seq). We identified two subgroups of NSCs: the subgroup located within the CC cannot prime to active NSCs after SCI, while the subgroup located outside the CC were activated and exhibited the active NSCs properties after SCI. We demonstrated the comprehensive dynamic transcriptome of NSCs from quiescent to active NSCs after SCI. This study reveals that Nestin+ cells outside CC were NSCs that activated upon SCI and may thus serve as endogenous NSCs for regenerative treatment of SCI in the future.

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Data availability The datasets generated and analyzed during the current study are available in the BIGD (National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences) database under the bioproject accession code: PRJCA003115.

References

  • Arai, Y., and Taverna, E. (2017). Neural progenitor cell polarity and cortical development. Front Cell Neurosci 11, 384.

    Article  PubMed  PubMed Central  Google Scholar 

  • Barnabé-Heider, F., Göritz, C., Sabelström, H., Takebayashi, H., Pfrieger, F.W., Meletis, K., and Frisén, J. (2010). Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell 7, 470–482.

    Article  PubMed  Google Scholar 

  • Becker, C.G., Becker, T., and Hugnot, J.P. (2018). The spinal ependymal zone as a source of endogenous repair cells across vertebrates. Prog Neurobiol 170, 67–80.

    Article  CAS  PubMed  Google Scholar 

  • Beckervordersandforth, R., Ebert, B., Schäffner, I., Moss, J., Fiebig, C., Shin, J., Moore, D.L., Ghosh, L., Trinchero, M.F., Stockburger, C., et al. (2017). Role of mitochondrial metabolism in the control of early lineage progression and aging phenotypes in adult hippocampal neurogenesis. Neuron 93, 560–573.e6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bernal, A., and Arranz, L. (2018). Nestin-expressing progenitor cells: function, identity and therapeutic implications. Cell Mol Life Sci 75, 2177–2195.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Butler, A., Hoffman, P., Smibert, P., Papalexi, E., and Satija, R. (2018). Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411–420.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cawsey, T., Duflou, J., Weickert, C.S., and Gorrie, C.A. (2015). Nestin-positive ependymal cells are increased in the human spinal cord after traumatic central nervous system injury. J Neurotrauma 32, 1393–1402.

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen, R., Wu, X., Jiang, L., and Zhang, Y. (2017). Single-cell RNA-seq reveals hypothalamic cell diversity. Cell Rep 18, 3227–3241.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Delile, J., Rayon, T., Melchionda, M., Edwards, A., Briscoe, J., and Sagner, A. (2019). Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord. Development 146, dev173807.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Devaraju, K., Barnabé-Heider, F., Kokaia, Z., and Lindvall, O. (2013). FoxJ1-expressing cells contribute to neurogenesis in forebrain of adult rats: Evidence from in vivo electroporation combined with piggyBac transposon. Exp Cell Res 319, 2790–2800.

    Article  CAS  PubMed  Google Scholar 

  • Dulken, B.W., Leeman, D.S., Boutet, S.C., Hebestreit, K., and Brunet, A. (2017). Single-cell transcriptomic analysis defines heterogeneity and transcriptional dynamics in the adult neural stem cell lineage. Cell Rep 18, 777–790.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fietz, S.A., and Huttner, W.B. (2011). Cortical progenitor expansion, self-renewal and neurogenesis—A polarized perspective. Curr Opin NeuroBiol 21, 23–35.

    Article  CAS  PubMed  Google Scholar 

  • Fu, X., Wu, S., Li, B., Xu, Y., and Liu, J. (2019). Functions of p53 in pluripotent stem cells. Protein Cell 11, 71–78.

    Article  PubMed  PubMed Central  Google Scholar 

  • Ghazale, H., Ripoll, C., Leventoux, N., Jacob, L., Azar, S., Mamaeva, D., Glasson, Y., Calvo, C.F., Thomas, J.L., Meneceur, S., et al. (2019). RNA profiling of the human and mouse spinal cord stem cell niches reveals an embryonic-like regionalization with MSX1+ roof-plate-derived cells. Stem Cell Rep 12, 1159–1177.

    Article  CAS  Google Scholar 

  • Göritz, C., Dias, D.O., Tomilin, N., Barbacid, M., Shupliakov, O., and Frisén, J. (2011). A pericyte origin of spinal cord scar tissue. Science 333, 238–242.

    Article  PubMed  Google Scholar 

  • Grün, D., and van Oudenaarden, A. (2015). Design and analysis of single-cell sequencing experiments. Cell 163, 799–810.

    Article  PubMed  Google Scholar 

  • Habib, N., Li, Y., Heidenreich, M., Swiech, L., Avraham-Davidi, I., Trombetta, J.J., Hession, C., Zhang, F., and Regev, A. (2016). Div-seq: single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science 353, 925–928.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hamilton, L.K., Truong, M.K.V., Bednarczyk, M.R., Aumont, A., and Fernandes, K.J.L. (2009). Cellular organization of the central canal ependymal zone, a niche of latent neural stem cells in the adult mammalian spinal cord. Neuroscience 164, 1044–1056.

    Article  CAS  PubMed  Google Scholar 

  • Hara, M., Kobayakawa, K., Ohkawa, Y., Kumamaru, H., Yokota, K., Saito, T., Kijima, K., Yoshizaki, S., Harimaya, K., Nakashima, Y., et al. (2017). Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury. Nat Med 23, 818–828.

    Article  CAS  PubMed  Google Scholar 

  • Hugnot, J.P., and Franzen, R. (2011). The spinal cord ependymal region: a stem cell niche in the caudal central nervous system. Front Biosci 16, 1044–1059.

    Article  CAS  Google Scholar 

  • Jacquet, B.V., Salinas-Mondragon, R., Liang, H., Therit, B., Buie, J.D., Dykstra, M., Campbell, K., Ostrowski, L.E., Brody, S.L., and Ghashghaei, H.T. (2009). FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain. Development 136, 4021–4031.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klein, T., Ling, Z., Heimberg, H., Madsen, O.D., Heller, R.S., and Serup, P. (2003). Nestin is expressed in vascular endothelial cells in the adult human pancreas. J Histochem Cytochem 51, 697–706.

    Article  CAS  PubMed  Google Scholar 

  • Li, X., Zhao, Y., Cheng, S., Han, S., Shu, M., Chen, B., Chen, X., Tang, F., Wang, N., Tu, Y., et al. (2017). Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair. Biomaterials 137, 73–86.

    Article  CAS  PubMed  Google Scholar 

  • Li, X., Floriddia, E.M., Toskas, K., Chalfouh, C., Honore, A., Aumont, A., Vallières, N., Lacroix, S., Fernandes, K.J.L., Guérout, N., et al. (2018a). FoxJ1 regulates spinal cord development and is required for the maintenance of spinal cord stem cell potential. Exp Cell Res 368, 84–100.

    Article  CAS  PubMed  Google Scholar 

  • Li, X., Fan, C., Xiao, Z., Zhao, Y., Zhang, H., Sun, J., Zhuang, Y., Wu, X., Shi, J., Chen, Y., et al. (2018b). A collagen microchannel scaffold carrying paclitaxel-liposomes induces neuronal differentiation of neural stem cells through Wnt/β-catenin signaling for spinal cord injury repair. Biomaterials 183, 114–127.

    Article  CAS  PubMed  Google Scholar 

  • Li, X., Liu, D., Xiao, Z., Zhao, Y., Han, S., Chen, B., and Dai, J. (2019). Scaffold-facilitated locomotor improvement post complete spinal cord injury: Motor axon regeneration versus endogenous neuronal relay formation. Biomaterials 197, 20–31.

    Article  CAS  PubMed  Google Scholar 

  • Llorens-Bobadilla, E., Zhao, S., Baser, A., Saiz-Castro, G., Zwadlo, K., and Martin-Villalba, A. (2015). Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury. Cell Stem Cell 17, 329–340.

    Article  CAS  PubMed  Google Scholar 

  • Luo, Y., Coskun, V., Liang, A., Yu, J., Cheng, L., Ge, W., Shi, Z., Zhang, K., Li, C., Cui, Y., et al. (2015). Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells. Cell 161, 1175–1186.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Matsumata, M., Sakayori, N., Maekawa, M., Owada, Y., Yoshikawa, T., and Osumi, N. (2012). The effects of Fabp7 and Fabp5 on postnatal hippocampal neurogenesis in the mouse. Stem Cell 30, 1532–1543.

    Article  CAS  Google Scholar 

  • Meletis, K., Barnabé-Heider, F., Carlén, M., Evergren, E., Tomilin, N., Shupliakov, O., and Frisén, J. (2008). Spinal cord injury reveals multilineage differentiation of ependymal cells. PLoS Biol 6, e182.

    Article  PubMed  PubMed Central  Google Scholar 

  • Mirzadeh, Z., Merkle, F.T., Soriano-Navarro, M., Garcia-Verdugo, J.M., and Alvarez-Buylla, A. (2008). Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3, 265–278.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muthusamy, N., Vijayakumar, A., Cheng Jr, G., and Ghashghaei, H.T. (2014). A knock-in Foxj1CreERT2::GFP mouse for recombination in epithelial cells with motile cilia. Genesis 52, 350–358.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muthusamy, N., Brumm, A., Zhang, X., Carmichael, S.T., and Ghashghaei, H.T. (2018). Foxj1 expressing ependymal cells do not contribute new cells to sites of injury or stroke in the mouse forebrain. Sci Rep 8, 1766.

    Article  PubMed  PubMed Central  Google Scholar 

  • Nomura, T., Göritz, C., Catchpole, T., Henkemeyer, M., and Frisén, J. (2010). EphB signaling controls lineage plasticity of adult neural stem cell niche cells. Cell Stem Cell 7, 730–743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Paniagua-Torija, B., Norenberg, M., Arevalo-Martin, A., Carballosa-Gautam, M.M., Campos-Martin, Y., Molina-Holgado, E., and Garcia-Ovejero, D. (2018). Cells in the adult human spinal cord ependymal region do not proliferate after injury. J Pathol 246, 415–421.

    Article  CAS  PubMed  Google Scholar 

  • Petit, A., Sanders, A.D., Kennedy, T.E., Tetzlaff, W., Glattfelder, K.J., Dalley, R.A., Puchalski, R.B., Jones, A.R., and Roskams, A.J. (2011). Adult spinal cord radial glia display a unique progenitor phenotype. PLoS ONE 6, e24538.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Picelli, S., Faridani, O.R., Björklund, A.K., Winberg, G., Sagasser, S., and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181.

    Article  CAS  PubMed  Google Scholar 

  • Ren, Y., Ao, Y., O’Shea, T.M., Burda, J.E., Bernstein, A.M., Brumm, A.J., Muthusamy, N., Ghashghaei, H.T., Carmichael, S.T., Cheng, L., et al. (2017). Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury. Sci Rep 7, 41122.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sabelström, H., Stenudd, M., and Frisén, J. (2014). Neural stem cells in the adult spinal cord. Exp Neurol 260, 44–49.

    Article  PubMed  Google Scholar 

  • Sakaguchi, M., Shingo, T., Shimazaki, T., Okano, H.J., Shiwa, M., Ishibashi, S., Oguro, H., Ninomiya, M., Kadoya, T., Horie, H., et al. (2006). A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells. Proc Natl Acad Sci USA 103, 7112–7117.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sakaguchi, M., Imaizumi, Y., and Okano, H. (2007). Expression and function of galectin-1 in adult neural stem cells. Cell Mol Life Sci 64, 1254–1258.

    Article  CAS  PubMed  Google Scholar 

  • Santra, M., Chopp, M., Zhang, Z.G., Lu, M., Santra, S., Nalani, A., Santra, S., and Morris, D.C. (2012). Thymosin beta4 mediates oligodendrocyte differentiation by upregulating p38 MAPK. Glia 60, 1826–1838.

    Article  PubMed  PubMed Central  Google Scholar 

  • Senturk, E., and Manfredi, J.J. (2013). p53 and cell cycle effects after DNA damage. In:Deb, S., and Deb, S., eds. p53 Protocols. Methods in Molecular Biology (Methods and Protocols). Totowa: Humana Press. 49–61.

    Google Scholar 

  • Shah, P.T., Stratton, J.A., Stykel, M.G., Abbasi, S., Sharma, S., Mayr, K.A., Koblinger, K., Whelan, P.J., and Biernaskie, J. (2018). Single-cell transcriptomics and fate mapping of ependymal cells reveals an absence of neural stem cell function. Cell 173, 1045–1057.e9.

    Article  CAS  PubMed  Google Scholar 

  • Shen, H., Wu, S., Chen, X., Xu, B., Ma, D., Zhao, Y., Zhuang, Y., Chen, B., Hou, X., Li, J., et al. (2020). Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: Long-term survival and functional recovery in canines. Sci China Life Sci 63, 1879–1886.

    Article  CAS  PubMed  Google Scholar 

  • Shin, J., Berg, D.A., Zhu, Y., Shin, J.Y., Song, J., Bonaguidi, M.A., Enikolopov, G., Nauen, D.W., Christian, K.M., Ming, G., et al. (2015). Single-cell RNA-seq with waterfall reveals molecular cascades underlying adult neurogenesis. Cell Stem Cell 17, 360–372.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck III, W.M., Hao, Y., Stoeckius, M., Smibert, P., and Satija, R. (2019). Comprehensive integration of single-cell data. Cell 177, 1888–1902.e21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Trapnell, C., Cacchiarelli, D., Grimsby, J., Pokharel, P., Li, S., Morse, M., Lennon, N.J., Livak, K.J., Mikkelsen, T.S., and Rinn, J.L. (2014). The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol 32, 381–386.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang, F., Flanagan, J., Su, N., Wang, L.C., Bui, S., Nielson, A., Wu, X., Vo, H.T., Ma, X.J., and Luo, Y. (2012). RNAscope. J Mol Diagn 14, 22–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu, X., Duan, S., Yi, F., Ocampo, A., Liu, G.H., and Izpisua Belmonte, J.C. (2013). Mitochondrial regulation in pluripotent stem cells. Cell Metab 18, 325–332.

    Article  CAS  PubMed  Google Scholar 

  • Xue, X., Shu, M., Xiao, Z., Zhao, Y., Li, X., Zhang, H., Fan, Y., Wu, X., Chen, B., Xu, B., et al. (2021). Lineage tracing reveals the origin of Nestin-positive cells are heterogeneous and rarely from ependymal cells after spinal cord injury. Sci China Life Sci doi: https://doi.org/10.1007/s11427-020-1901-4.

  • Yamada, J., and Jinno, S. (2014). S100A6 (calcyclin) is a novel marker of neural stem cells and astrocyte precursors in the subgranular zone of the adult mouse hippocampus. Hippocampus 24, 89–101.

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi, M., Saito, H., Suzuki, M., and Mori, K. (2000). Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice. Neuroreport 11, 1991–1996.

    Article  CAS  PubMed  Google Scholar 

  • Yu, G., Wang, L.G., Han, Y., and He, Q.Y. (2012). clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu, X., Ng, C.P., Habacher, H., and Roy, S. (2008). Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat Genet 40, 1445–1453.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81891002 and 81891001), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16040700), the National Key Research and Development Program of China (2017YFA0104701, 2017YFA0104704 and 2016YFC1101501), and Jiangsu Key Research and Development Program (BE2018664).

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  1. Contributed equally to this work

Authors and Affiliations

  1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100080, China

    Muya Shu, Xiaoyu Xue, Hu Nie, Xianming Wu, Minghan Sun, Lianyong Qiao, Bai Xu, Zhifeng Xiao, Yannan Zhao, Yongheng Fan, Bing Chen, Jixiang Zhang, Ya Shi, Yaming Yang, Falong Lu & Jianwu Dai

  2. University of the Chinese Academy of Sciences, Beijing, 100101, China

    Falong Lu & Jianwu Dai

  3. Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, 410008, China

    Xing Li

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  1. Muya Shu
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Correspondence to Falong Lu or Jianwu Dai.

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Shu, M., Xue, X., Nie, H. et al. Single-cell RNA sequencing reveals Nestin+ active neural stem cells outside the central canal after spinal cord injury. Sci. China Life Sci. 65, 295–308 (2022). https://doi.org/10.1007/s11427-020-1930-0

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