Brain Research 874 (2000) 87–106 www.elsevier.com / locate / bres Research report Characterization and intraspinal grafting of EGF / bFGF-dependent neurospheres derived from embryonic rat spinal cord a, a a b a * Stella Y. Chow , Jon Moul , Chris A. Tobias , B. Timothy Himes , Yi Liu , a a b a Maria Obrocka , Lisa Hodge , Alan Tessler , Itzhak Fischer a Department of Neurobiology and Anatomy, MCP Hahnemann University, 3200 Henry Avenue, Philadelphia, PA 19129, USA b Department of Veteran Affairs Medical Center, Philadelphia, PA 19104, USA Accepted 25 April 2000 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-immuno- suppressed 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.  2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Transplantation Keywords: Precursor; Progenitor; Stem cell; Transplantation; BDNF; Retinoic acid 1. Introduction Transplants of fetal CNS tissue or peripheral nerve, however, induce little regeneration into the host and permit Intraspinal grafts of fetal tissue, peripheral nerve, pri- only limited functional recovery. Optimal repair and mary cells or cell lines have been used in experiments to recovery after CNS injury may require combinations of repair spinal cord injury. Grafts can act as a bridge for different factors to stimulate axonal growth and protect regenerating host axons, transplanted neurons can act as a injured neurons. These factors may include neurotrophins relay between regenerating host axons and denervated host to stimulate axonal sprouting and elongation and to protect neurons, and molecules presented by transplanted tissue injured neurons, molecules that will neutralize axonal can be neuroprotective, rescuing host neurons that would growth inhibitors, provide a permissive extracellular en- otherwise die [60,61]. The trophic influences provided by vironment, and ameliorate the toxic environment at the transplanted cells may stimulate regenerative sprouting, lesion site. diminish the immune response and reduce the glial scar. 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, cul- *Corresponding author. Tel.: 11-215-842-4635; fax: 11-215-843- tured cells are genetically modified to express the neces- 9082. E-mail address: chow@drexel.edu (S.Y. Chow). sary therapeutic gene products, such as neurotrophins, and 0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993(00)02443-4