Research reportCharacterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord
Introduction
Intraspinal grafts of fetal tissue, peripheral nerve, primary cells or cell lines have been used in experiments to repair spinal cord injury. Grafts can act as a bridge for regenerating host axons, transplanted neurons can act as a relay between regenerating host axons and denervated host neurons, and molecules presented by transplanted tissue can be neuroprotective, rescuing host neurons that would otherwise die [60], [61]. The trophic influences provided by transplanted cells may stimulate regenerative sprouting, diminish the immune response and reduce the glial scar. Transplants of fetal CNS tissue or peripheral nerve, however, induce little regeneration into the host and permit only limited functional recovery. Optimal repair and recovery after CNS injury may require combinations of different factors to stimulate axonal growth and protect injured neurons. These factors may include neurotrophins to stimulate axonal sprouting and elongation and to protect injured neurons, molecules that will neutralize axonal growth inhibitors, provide a permissive extracellular environment, and ameliorate the toxic environment at the lesion site.
Ex vivo gene therapy is a promising approach for improving spinal cord grafts since cells can be modified to supply factors needed for repair. With this strategy, cultured cells are genetically modified to express the necessary therapeutic gene products, such as neurotrophins, and then transplanted into the injury site where they can act both as a source of factors that support repair and as a bridge for regenerating host axons. Recently, intraspinal transplants of genetically modified primary fibroblasts have been shown to induce regeneration of host axons, as well as improving functional recovery after spinal cord injury [16], [36]. Transplants of cells that can be genetically modified and also differentiate into neural cell types offer the same advantages that fibroblasts provide but in addition offer a potential for cellular replacement.
CNS stem cells are defined by their ability to proliferate, self-renew, and retain the potential to generate progenitor cells that can differentiate into neurons and glia [30], [38], [47], [65], [66]. Progenitor cells have a more restricted lineage, differentiating into either neurons or glia. Precursor cells include both stem cells and progenitor cells [14], [55]. Multipotent neural stem cells have been isolated from both the embryonic and adult brain [6], [7], [27], [40], [48], [50], [63] and spinal cord [46], [64]. Treatment of these cells in vitro with specific growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or transforming growth factor-α (TGF-α) allows them to remain in the proliferative state [14]. During development, terminally differentiated neurons and glia are generated from multipotent stem cells [59]. The signals that regulate the fate and lineage commitment of these cells differ according to the specific region and developmental stage of the nervous system [4]. Such signals can have selective effects on the survival or proliferation of a subpopulation of progenitor cells, as well as instructive effects on phenotypic outcome [30], [31], [47], [58]. For example, neural stem cells from adult mouse striatum survive and proliferate in response to EGF alone [49] or bFGF alone [17], but neural stem cells from adult mouse spinal cord only survive and proliferate when exposed to both EGF and bFGF [64]. EGF-responsive striatal stem cells do not proliferate in response to bFGF alone [49] but, when exposed to bFGF and fetal calf serum (FCS), they generate two populations of progenitor cells: a unipotent neuronal progenitor and a bipotent neuronal/astrocytic progenitor [62]. Extrinsic factors also affect phenotypic choice. Embryonic striatal cells expanded either with EGF or bFGF give rise to neurons, astrocytes, or oligodendrocytes but more astrocytes are generated when cells are expanded in the presence of EGF alone. Subsequent exposure to PDGF almost doubles the percent of bFGF-expanded cells that differentiate into neurons, while EGF-expanded cells show only a minimal increase in neuronal differentiation [24]. Optimal conditions may therefore need to be identified for each CNS region. Grafting experiments have, however, demonstrated that once these precursor cells are transplanted, many of them can respond to local environmental cues by differentiating into region appropriate cells [12].
Neural stem or progenitor cells that can be maintained in vitro in an actively proliferating state while maintaining the capacity to differentiate into mature neurons and glia are attractive candidates for use as transplants to repair the damaged CNS. The potential to produce in vitro the desired proportions of glial or neuronal progenitors using defined extrinsic factors allows the design of cellular transplants that can fulfil specific needs in the repair of the damaged CNS.
In this study we examined the proliferation and growth of embryonic rat spinal cord cells isolated in the presence of EGF and bFGF and the induction of glial and neuronal phenotypes by extrinsic factors in vitro. We then evaluated the potential of these cells as an intraspinal transplant by examining their survival and differentiation after grafting into the injured spinal cord. We found that cells isolated from the spinal cord of embryonic Sprague–Dawley and Fischer 344 rats proliferate in the presence of EGF and bFGF, can be expanded for multiple passages, and have the capacity to differentiate into neurons and glia in vitro. These cells show promise as intraspinal grafts because they survive well in injured spinal cord, differentiate into multiple cell types in vivo, are permissive for host axon growth, and are easily modified by adenoviral vectors.
Section snippets
Isolation and expansion of embryonic spinal cord cells
Rat embryonic day 14 (E14) spinal cord (Sprague–Dawley or Fischer 344 rats from Taconic Farms) was dissected in DMEM medium. The tissue was rinsed in Hank’s buffered saline solution (HBSS), cut into small pieces and transferred into full growth media composed of DMEM/F12 (1:1), HEPES buffer (5 mM), glucose (0.6%), sodium bicarbonate (3 mM), glutamine (2 mM), EGF (20 ng/ml, Collaborative Research), bFGF (20 ng/ml, Collaborative Research) and a defined hormone and salt mixture composed of insulin
Isolation, expansion, and storage of embryonic spinal cord cells
E14 spinal cords were dissociated and plated in medium containing bFGF and EGF. The dissociated spinal cord cells rapidly proliferated and the dividing cells aggregated and formed free-floating spheres (Fig. 1A). These spheres resembled the neurospheres described previously for adult murine forebrain subependymal and spinal cord stem cells [49], [64]. The majority of cells within the sphere expressed the intermediate filament protein, nestin, a marker for neural precursor cells [69], [70] (Fig.
Discussion
We show that spinal cord cells isolated from embryonic day 14 rats and grown in the presence of EGF and bFGF, proliferate as undifferentiated cells and can be expanded over long periods of time. When induced to differentiate by extrinsic factors, they can become neurons, astrocytes, or oligodendrocytes. These cells survive when grafted into the injured spinal cord and therefore can be used as an intraspinal transplant in models of spinal cord injury. They are easily modified by adenoviral
Acknowledgements
This work was supported by National Institutes of Health Grant NS24707 and Training Grants NS10090 and HD07467, The Spinal Cord Research Foundation of the Paralyzed Veterans Association, The Eastern Paralyzed Veterans Association, the International Spinal Cord Research Trust, a Center of Excellence Grant from Medical College of Pennsylvania/Hahnemann University, and the Research Service of the Department of Veteran Affairs. We thank Dr. Marion Murray for her suggestions and critical comments on
References (70)
Intrinsic and extrinsic factors regulating vertebrate neurogenesis
Curr. Opin. Neurobiol.
(1995)- et al.
Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors
Neuron
(1994) - et al.
Regulation of microtubule associated protein 2 (MAP2) expression by nerve growth factor in PC12 cells
Exp. Cell Res.
(1991) Neural precursor cells: applications for the study and repair of the central nervous system
Neurobiol. Dis.
(1997)- et al.
Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis
Neuron
(1995) - et al.
Identification of a neural stem cell in the adult mammalian central nervous system
Cell
(1999) - et al.
Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis
Dev. Biol.
(1997) - et al.
Embryonic precursor cells that express trk receptors: induction of different cell fates by NGF, BDNF, NT-3 and CNTF
Exp. Neurol.
(1997) Neural progenitors and stem cells: mechanisms of progenitor heterogeneity
Curr. Opin. Neurobiol.
(1998)- et al.
Application of recombinant adenovirus for in vivo gene delivery to spinal cord
Brain Res.
(1997)
Intraspinal delivery of neurotrophin-3 (NT-3) using neural stem cells genetically modified by recombinant retrovirus
Exp. Neurol.
Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells
Neuron
Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells
Neuron
The adult rat hippocampus contains primordial neural stem cells
Mol. Cell. Neurosci.
Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell
Dev. Biol.
Neural stem cells are blasting off
Neuron
Restricted growth potential of rat neural precursors as compared to mouse
Dev. Brain Res.
Stem cells in the adult mammalian central nervous system
Curr. Opin. Neurobiol.
Vertebrate neural progenitor cells: subtypes and regulation
Curr. Opin. Neurobiol.
bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells
Neuron
Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons
Neuron
Retinoids increase perinatal spinal cord neuronal survival and astroglial differentiation
Int. J. Dev. Neurosci.
Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons
Exp. Neurol.
CNS stem cells express a new class of intermediate filament protein
Cell
BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors
J. Neurosci.
Microtubule-associated protein 1b (MAP 1b) is concentrated in the distal region of growing axons
J. Neurosci.
NT-3, but not BDNF, prevents atrophy and death of axotomized spinal cord projection neurons
Eur. J. Neurosci.
Isografts of neural stem cells into spinal cord of Fischer 344 rats
Soc. Neurosci. Abstr.
A self-renewing multipotential stem cell in embryonic rat cerebral cortex
Nature
Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain
J. Neurosci.
Rapid, widespread, and longstanding induction of nestin contributes to the generation of glial scar tissue after CNS injury
J. Cell Biol.
Mammalian neural stem cells
Science
Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain
Proc. Natl. Acad. Sci. USA
Isolation, characterization, and use of stem cells from the CNS
Annu. Rev. Neurosci.
Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury
J. Neurosci.
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