Grafted Lineage-Restricted Precursors Differentiate Exclusively into Neurons in the Adult Spinal Cord

Multipotent neural stem cells (NSCs) have the potential to differentiate into neuronal and glial cells and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages. In particular, when grafted into the adult spinal cord, NSCs are restricted to the glial lineage, indicating that the host spinal cord environment is not permissive for neuronal differentiation. To identify the stage at which neuronal differentiation is inhibited we examined the survival, differentiation, and integration of neuronal restricted precursor (NRP) cells, derived from the embryonic spinal cord of transgenic alkaline phosphatase rats, after transplantation into the adult spinal cord. We found that grafted NRP cells differentiate into mature neurons, survive for at least 1 month, appear to integrate within the host spinal cord, and extend processes in both the gray and white matter. Conversely, grafted glial restricted precursor cells did not differentiate into neurons. We did not observe glial differentiation from the grafted NRP cells, indicating that they retained their neuronal restricted properties in vivo. We conclude that the adult nonneurogenic CNS environment does not support the transition of multipotential NSCs to the neuronal commitment stage, but does allow the survival, maturation, and integration of NRP cells.

[1]  P. Horner,et al.  Adult Spinal Cord Stem Cells Generate Neurons after Transplantation in the Adult Dentate Gyrus , 2000, The Journal of Neuroscience.

[2]  H. Keirstead,et al.  Stem cell transplantation into the central nervous system and the control of differentiation , 2001, Journal of neuroscience research.

[3]  F. Gage,et al.  Proliferation, differentiation, and long-term culture of primary hippocampal neurons. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Steindler,et al.  Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  I. Fischer,et al.  Isolation of a glial‐restricted tripotential cell line from embryonic spinal cord cultures , 2002, Glia.

[6]  S. Dunnett,et al.  Survival and Differentiation of Rat and Human Epidermal Growth Factor-Responsive Precursor Cells Following Grafting into the Lesioned Adult Central Nervous System , 1996, Experimental Neurology.

[7]  R. J. Mullen,et al.  NeuN, a neuronal specific nuclear protein in vertebrates. , 1992, Development.

[8]  J. García-Verdugo,et al.  Adult‐derived neural precursors transplanted into multiple regions in the adult brain , 1999, Annals of neurology.

[9]  Sally Temple,et al.  The development of neural stem cells , 2001, Nature.

[10]  Bertram Wiedenmann,et al.  Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles , 1985, Cell.

[11]  R. McKay,et al.  Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. , 1996, Genes & development.

[12]  J. D. Macklis,et al.  Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Raisman,et al.  Long interfascicular axon growth from embryonic neurons transplanted into adult myelinated tracts , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[14]  S. Temple,et al.  A self-renewing multipotential stem cell in embryonic rat cerebral cortex , 1994, Nature.

[15]  Arturo Alvarez-Buylla,et al.  A unified hypothesis on the lineage of neural stem cells , 2001, Nature Reviews Neuroscience.

[16]  S. Weiss,et al.  Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. , 1992, Science.

[17]  J. Trojanowski,et al.  Differential effects of spinal cord gray and white matter on process outgrowth from grafted human NTERA2 neurons (NT2N, hNT) , 1999, The Journal of comparative neurology.

[18]  Neural stem cells and gene therapy: prospects for repairing the injured spinal cord. , 2000, JAMA.

[19]  Scott R. Whittemore,et al.  Pluripotent Stem Cells Engrafted into the Normal or Lesioned Adult Rat Spinal Cord Are Restricted to a Glial Lineage , 2001, Experimental Neurology.

[20]  G. Raisman,et al.  Long Fibre Growth by Axons of Embryonic Mouse Hippocampal Neurons Microtransplanted into the Adult Rat Fimbria , 1993, The European journal of neuroscience.

[21]  I. Fischer,et al.  Stable Expression of the Alkaline Phosphatase Marker Gene by Neural Cells in Culture and after Transplantation into the CNS Using Cells Derived from a Transgenic Rat , 2002, Experimental Neurology.

[22]  P. Tresco,et al.  Embryonic-Derived Glial-Restricted Precursor Cells (GRP Cells) Can Differentiate into Astrocytes and Oligodendrocytes in Vivo , 2001, Experimental Neurology.

[23]  M. Rao,et al.  Spinal Cord Neuronal Precursors Generate Multiple Neuronal Phenotypes in Culture , 1998, The Journal of Neuroscience.

[24]  Charles Tator,et al.  Cell proliferation and nestin expression in the ependyma of the adult rat spinal cord after injury. , 1999, Journal of neuropathology and experimental neurology.

[25]  A. Tessler,et al.  Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord , 2000, Brain Research.

[26]  R. McKay,et al.  Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells , 2000, Nature Biotechnology.

[27]  B. Stokes,et al.  Proliferation of NG2-Positive Cells and Altered Oligodendrocyte Numbers in the Contused Rat Spinal Cord , 2001, The Journal of Neuroscience.

[28]  Kozo Nakamura,et al.  Transcription Factor Expression and Notch-Dependent Regulation of Neural Progenitors in the Adult Rat Spinal Cord , 2001, The Journal of Neuroscience.

[29]  M. Murray,et al.  Intraspinal Delivery of Neurotrophin-3 Using Neural Stem Cells Genetically Modified by Recombinant Retrovirus , 1999, Experimental Neurology.

[30]  M. Rao,et al.  Cell lineage in the developing neural tube. , 1998, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[31]  V. P. Collins,et al.  Immunohistochemistry of a spontaneous murine ovarian teratoma with neuroepithelial differentiation. Neuron-associated beta-tubulin as a marker for primitive neuroepithelium. , 1989, Laboratory investigation; a journal of technical methods and pathology.

[32]  F. Gage,et al.  Mammalian neural stem cells. , 2000, Science.

[33]  B. Walker,et al.  INCORPORATION OF THYMIDINE‐H3 BY CELLS IN NORMAL AND INJURED MOUSE SPINAL CORD , 1962, Journal of neuropathology and experimental neurology.

[34]  A. Álvarez-Buylla,et al.  Stem cells in the adult mammalian central nervous system , 1999, Current Opinion in Neurobiology.

[35]  Thomas C. Südhof,et al.  The synaptic vesicle cycle: a cascade of protein–protein interactions , 1995, Nature.

[36]  G. Wilcox,et al.  Neurogenesis in postnatal rat spinal cord: a study in primary culture. , 1997, Science.

[37]  J. D. Macklis,et al.  Neural Precursor Differentiation Following Transplantation into Neocortex Is Dependent on Intrinsic Developmental State and Receptor Competence , 1999, Experimental Neurology.

[38]  L. Wysocki,et al.  "Panning" for lymphocytes: a method for cell selection. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[39]  E. K. Adrian.  Cell division in injured spinal cord , 1968 .

[40]  K. Hobson,et al.  Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis. , 1997, Developmental biology.

[41]  Jonas Frisén,et al.  Identification of a Neural Stem Cell in the Adult Mammalian Central Nervous System , 1999, Cell.

[42]  M. Raff,et al.  Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. , 2000, Science.

[43]  Daniel A. Lim,et al.  Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult Mammalian Brain , 1999, Cell.

[44]  M. Luskin,et al.  Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Stacey P. Memberg,et al.  Regeneration of adult axons in white matter tracts of the central nervous system , 1997, Nature.

[46]  M. Rao,et al.  Gliogenesis in the central nervous system , 2000, Glia.

[47]  F. Gage,et al.  Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo , 1996, Nature.

[48]  A. Matus.  Microtubule-associated proteins: their potential role in determining neuronal morphology. , 1988, Annual review of neuroscience.

[49]  Kozo Nakamura,et al.  Proliferation of Parenchymal Neural Progenitors in Response to Injury in the Adult Rat Spinal Cord , 2001, Experimental Neurology.

[50]  F. Gage,et al.  Proliferation and Differentiation of Progenitor Cells Throughout the Intact Adult Rat Spinal Cord , 2000, The Journal of Neuroscience.

[51]  F. Gage,et al.  Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[52]  M. Rao.  Stem cells and CNS development , 2001 .

[53]  W. Kisseberth,et al.  Ubiquitous expression of marker transgenes in mice and rats. , 1999, Developmental biology.

[54]  M. Rao,et al.  Glial-restricted precursors are derived from multipotent neuroepithelial stem cells. , 1997, Developmental biology.

[55]  C. Svendsen,et al.  Neural stem cells in the developing central nervous system: implications for cell therapy through transplantation. , 2000, Progress in brain research.

[56]  M. Rao,et al.  Precursor cells for transplantation. , 2000, Progress in brain research.

[57]  Scott R. Whittemore,et al.  Differentiation of Engrafted Neuronal-Restricted Precursor Cells Is Inhibited in the Traumatically Injured Spinal Cord , 2002, Experimental Neurology.

[58]  M. Segal,et al.  Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro , 1996, Mechanisms of Development.

[59]  M. Rao,et al.  Isolation of Lineage-Restricted Neuronal Precursors from Multipotent Neuroepithelial Stem Cells , 1997, Neuron.