Advertisement

Cellular and Molecular Neurobiology

, Volume 35, Issue 1, pp 57–70 | Cite as

Time Course of Spinal Doublecortin Expression in Developing Rat and Porcine Spinal Cord: Implication in In Vivo Neural Precursor Grafting Studies

  • J. Juhasova
  • S. Juhas
  • M. Hruska-Plochan
  • D. Dolezalova
  • M. Holubova
  • J. Strnadel
  • S. Marsala
  • J. Motlik
  • M. Marsala
Original Research

Abstract

Expression of doublecortin (DCX), a 43–53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113–114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70–80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3–4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

Keywords

Doublecortin Spinal cord development Spinal neural precursor grafting Minipig Rat GFAP 

Notes

Acknowledgments

This study was funded by CIRM (M.M.), TA01011466, CZ.1.05./2.1.00/03.0124, and RVO: 67985904 (J.J., S.J., J.M., J.S., M.H.).

References

  1. Cao QL, Onifer SM, Whittemore SR (2008) Labeling stem cells in vitro for identification of their differentiated phenotypes after grafting into the CNS. Methods Mol Biol 438:361–374PubMedCrossRefGoogle Scholar
  2. Cizkova D, Rosocha J, Vanicky I, Jergova S, Cizek M (2006) Transplants of human mesenchymal stem cells improve functional recovery after spinal cord injury in the rat. Cell Mol Neurobiol 26(7–8):1165–1178CrossRefGoogle Scholar
  3. Cizkova D, Kakinohana O, Kucharova K, Marsala S, Johe K, Hazel T, Hefferan MP, Marsala M (2007) Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells. Neuroscience 147(2):546–560. doi: 10.1016/j.neuroscience.2007.02.065 PubMedCentralPubMedCrossRefGoogle Scholar
  4. Couillard-Despres S, Winner B, Schaubeck S, Aigner R, Vroemen M, Weidner N, Bogdahn U, Winkler J, Kuhn HG, Aigner L (2005) Doublecortin expression levels in adult brain reflect neurogenesis. Eur J Neurosci 21(1):1–14PubMedCrossRefGoogle Scholar
  5. Couillard-Despres S, Finkl R, Winner B, Ploetz S, Wiedermann D, Aigner R, Bogdahn U, Winkler J, Hoehn M, Aigner L (2008) In vivo optical imaging of neurogenesis: watching new neurons in the intact brain. Mol Imaging 7(1):28–34PubMedGoogle Scholar
  6. Darsalia V, Kallur T, Kokaia Z (2007) Survival, migration and neuronal differentiation of human fetal striatal and cortical neural stem cells grafted in stroke-damaged rat striatum. Eur J Neurosci 26(3):605–614PubMedCrossRefGoogle Scholar
  7. Dolezalova D, Hruska-Plochan M, Bjarkam CR, Sorensen JC, Cunningham M, Weingarten D, Ciacci JD, Juhas S, Juhasova J, Motlik J, Hefferan MP, Hazel T, Johe K, Carromeu C, Muotri A, Bui J, Strnadel J, Marsala M (2014) Pig models of neurodegenerative disorders: utilization in cell replacement-based preclinical safety and efficacy studies. J Comp Neurol. doi: 10.1002/cne.23575 PubMedGoogle Scholar
  8. Englund U, Bjorklund A, Wictorin K (2002) Migration patterns and phenotypic differentiation of long-term expanded human neural progenitor cells after transplantation into the adult rat brain. Brain Res Dev Brain Res 134(1–2):123–141PubMedCrossRefGoogle Scholar
  9. Gleeson JG, Lin PT, Flanagan LA, Walsh CA (1999) Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 23(2):257–271PubMedCrossRefGoogle Scholar
  10. Hakamata Y, Tahara K, Uchida H, Sakuma Y, Nakamura M, Kume A, Murakami T, Takahashi M, Takahashi R, Hirabayashi M, Ueda M, Miyoshi I, Kasai N, Kobayashi E (2001) Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun 286(4):779–785PubMedCrossRefGoogle Scholar
  11. Hefferan MP, Galik J, Kakinohana O, Sekerkova G, Santucci C, Marsala S, Navarro R, Hruska-Plochan M, Johe K, Feldman E, Cleveland DW, Marsala M (2012) Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation. PLoS One 7(8):e42614. doi: 10.1371/journal.pone.0042614 PubMedCentralPubMedCrossRefGoogle Scholar
  12. Hirano M, Goldman JE (1988) Gliogenesis in rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors. J Neurosci Res 21(2–4):155–167. doi: 10.1002/jnr.490210208 PubMedCrossRefGoogle Scholar
  13. Horner PJ, Power AE, Kempermann G, Kuhn HG, Palmer TD, Winkler J, Thal LJ, Gage FH (2000) Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J Neurosci 20(6):2218–2228PubMedGoogle Scholar
  14. Hua R, Doucette R, Walz W (2008) Doublecortin-expressing cells in the ischemic penumbra of a small-vessel stroke. J Neurosci Res 86(4):883–893PubMedCrossRefGoogle Scholar
  15. Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, Sato Y, Kaneko T, Takahashi M, Okada T, Ozawa K, Francis J, Leone P, Kobayashi E (2005) Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun 329(1):288–295PubMedCrossRefGoogle Scholar
  16. Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci USA 99(18):11946–11950PubMedCentralPubMedCrossRefGoogle Scholar
  17. Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisén J (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96(1):25–34PubMedCrossRefGoogle Scholar
  18. Kakinohana O, Cizkova D, Tomori Z, Hedlund E, Marsala S, Isacson O, Marsala M (2004) Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study. Exp Neurol 190(1):122–132. doi: 10.1016/j.expneurol.2004.07.014 PubMedCrossRefGoogle Scholar
  19. Kakinohana O, Juhasova J, Juhas S, Motlik J, Platoshyn O, Galik J, Hefferan M, Yuan SH, Vidal JG, Carson CT, van Gorp S, Goldberg D, Leerink M, Lazar P, Marsala S, Miyanohara A, Keshavarzi S, Ciacci JD, Marsala M (2012) Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study. Cell Transpl 21(12):2603–2619. doi: 10.3727/096368912X653200 CrossRefGoogle Scholar
  20. Karl C, Couillard-Despres S, Prang P, Munding M, Kilb W, Brigadski T, Plotz S, Mages W, Luhmann H, Winkler J, Bogdahn U, Aigner L (2005) Neuronal precursor-specific activity of a human doublecortin regulatory sequence. J Neurochem 92(2):264–282PubMedCrossRefGoogle Scholar
  21. Kawaguchi A, Miyata T, Sawamoto K, Takashita N, Murayama A, Akamatsu W, Ogawa M, Okabe M, Tano Y, Goldman SA, Okano H (2001) Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells. Mol Cell Neurosci 17(2):259–273PubMedCrossRefGoogle Scholar
  22. Kelly S, Bliss TM, Shah AK, Sun GH, Ma M, Foo WC, Masel J, Yenari MA, Weissman IL, Uchida N, Palmer T, Steinberg GK (2004) Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci USA 101(32):11839–11844PubMedCentralPubMedCrossRefGoogle Scholar
  23. Lepore AC, Fischer I (2005) Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord. Exp Neurol 194(1):230–242PubMedCrossRefGoogle Scholar
  24. Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D, Brock J, Blesch A, Rosenzweig ES, Havton LA, Zheng B, Conner JM, Marsala M, Tuszynski MH (2012) Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150(6):1264–1273. doi: 10.1016/j.cell.2012.08.020 PubMedCentralPubMedCrossRefGoogle Scholar
  25. Magavi SS, Macklis JD (2008) Immunocytochemical analysis of neuronal differentiation. Methods Mol Biol 438:345–352PubMedCrossRefGoogle Scholar
  26. Marchetto MC, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR (2010) A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143(4):527–539. doi: 10.1016/j.cell.2010.10.016 PubMedCentralPubMedCrossRefGoogle Scholar
  27. Marsala M, Kakinohana O, Yaksh TL, Tomori Z, Marsala S, Cizkova D (2004) Spinal implantation of hNT neurons and neuronal precursors: graft survival and functional effects in rats with ischemic spastic paraplegia. Eur J Neurosci 20(9):2401–2414. doi: 10.1111/j.1460-9568.2004.03702.x PubMedCrossRefGoogle Scholar
  28. Nacher J, Crespo C, McEwen BS (2001) Doublecortin expression in the adult rat telencephalon. Eur J Neurosci 14(4):629–644PubMedCrossRefGoogle Scholar
  29. Nornes HO, Das GD (1974) Temporal pattern of neurogenesis in spinal cord of rat. I. An autoradiographic study–time and sites of origin and migration and settling patterns of neuroblasts. Brain Res 73(1):121–138PubMedCrossRefGoogle Scholar
  30. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407(3):313–319PubMedCrossRefGoogle Scholar
  31. Oudega M, Marani E (1991) Expression of vimentin and glial fibrillary acidic protein in the developing rat spinal cord: an immunocytochemical study of the spinal cord glial system. J Anat 179:97–114PubMedCentralPubMedGoogle Scholar
  32. Sabelström H, Stenudd M, Frisén J (2014) Neural stem cells in the adult spinal cord. Exp Neurol 260:44–49. doi: 10.1016/j.expneurol.2013.01.026 PubMedCrossRefGoogle Scholar
  33. Schwartz PH, Nethercott H, Kirov II, Ziaeian B, Young MJ, Klassen H (2005) Expression of neurodevelopmental markers by cultured porcine neural precursor cells. Stem Cells 23(9):1286–1294. doi: 10.1634/stemcells.2004-0306 PubMedCrossRefGoogle Scholar
  34. Sevc J, Goldberg D, van Gorp S, Leerink M, Juhas S, Juhasova J, Marsala S, Hruska-Plochan M, Hefferan MP, Motlik J, Rypacek F, Machova L, Kakinohana O, Santucci C, Johe K, Lukacova N, Yamada K, Bui JD, Marsala M (2013) Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: effect on spinally grafted human neural precursor survival. Exp Neurol 248:85–99. doi: 10.1016/j.expneurol.2013.05.017 PubMedCrossRefGoogle Scholar
  35. Tabar V, Panagiotakos G, Greenberg ED, Chan BK, Sadelain M, Gutin PH, Studer L (2005) Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat Biotechnol 23(5):601–606PubMedCrossRefGoogle Scholar
  36. Usvald D, Vodicka P, Hlucilova J, Prochazka R, Motlik J, Strnadel J, Kucharova K, Johe K, Marsala S, Scadeng M, Kakinohana O, Navarro R, Santa M, Hefferan MP, Yaksh TL, Marsala M (2010) Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs. Cell Transpl 19:1103–1122. doi: 10.3727/096368910X503406 CrossRefGoogle Scholar
  37. van Gorp S, Leerink M, Kakinohana O, Platoshyn O, Santucci C, Galik J, Joosten EA, Hruska-Plochan M, Goldberg D, Marsala S, Johe K, Ciacci JD, Marsala M (2013) Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation. Stem Cell Res Ther 4(5):57. doi: 10.1186/scrt209 PubMedCentralPubMedCrossRefGoogle Scholar
  38. Walker TL, Yasuda T, Adams DJ, Bartlett PF (2007) The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells. J Neurosci 27(14):3734–3742PubMedCrossRefGoogle Scholar
  39. Wallace PK, Muirhead KA (2007) Cell tracking 2007: a proliferation of probes and applications. Immunol Invest 36(5–6):527–561PubMedCrossRefGoogle Scholar
  40. Yang H, Lu P, McKay HM, Bernot T, Keirstead H, Steward O, Gage FH, Edgerton VR, Tuszynski MH (2006) Endogenous neurogenesis replaces oligodendrocytes and astrocytes after primate spinal cord injury. J Neurosci 26(8):2157–2166. doi: 10.1523/JNEUROSCI.4070-05.2005 PubMedCrossRefGoogle Scholar
  41. Yuan SH, Martin J, Elia J, Flippin J, Paramban RI, Hefferan MP, Vidal JG, Mu Y, Killian RL, Israel MA, Emre N, Marsala S, Marsala M, Gage FH, Goldstein LS, Carson CT (2011) Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6(3):e17540. doi: 10.1371/journal.pone.0017540 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • J. Juhasova
    • 1
  • S. Juhas
    • 1
  • M. Hruska-Plochan
    • 2
    • 4
  • D. Dolezalova
    • 2
    • 5
  • M. Holubova
    • 3
  • J. Strnadel
    • 2
    • 3
  • S. Marsala
    • 2
  • J. Motlik
    • 1
  • M. Marsala
    • 2
  1. 1.Laboratory of Cell Regeneration and PlasticityInstitute of Animal Physiology and Genetics, AS CR, v.v.i.LibechovCzech Republic
  2. 2.Department of AnesthesiologyUniversity of California, San DiegoLa JollaUSA
  3. 3.Laboratory od Tumor BiologyInstitute of Animal Physiology and Genetics, AS CR, v.v.i.LibechovCzech Republic
  4. 4.Institute of Molecular Life SciencesUniversity of ZurichZurichSwitzerland
  5. 5.Department of Histology and Embryology, Faculty of MedicineMasaryk UniversityBrnoCzech Republic

Personalised recommendations