Elsevier

Brain Research

Volume 874, Issue 2, 25 August 2000, Pages 87-106
Brain Research

Research report
Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord

https://doi.org/10.1016/S0006-8993(00)02443-4Get rights and content

Abstract

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague–Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague–Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured 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

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