| Repair of Spinal cord injury is one of the most challenging issues in the present field of Neuroscience. In the past decade, basic science advances in spinal cord injury and regeneration research have led to a variety of novel experimental therapeutics designed to promote functionally effective axonal regrowth and sprouting. Before they are used to treat patients with spinal cord injuries, there are many issues to be considered relative to treatment safety and efficacy. A more readily accessible source of human cells, preferably with the potential for autologous transplantation, is needed.The olfactory neuroepithelium (ONe) undergoes life-long repair through the action of endogenous progenitors that replace its' neuronal and supporting cells. The ONe, by virtue of its accessible location, has special advantages as a source of CNS progenitors for autologous transplantation. In the last several years, our laboratory has developed endoscopic biopsy and culture procedures for obtaining human adult ONe-derived progenitors (ONPs). These progenitors can be removed from the primary cultures following their formation of neurospheres.The neurosphere forming cells (NSFCs) have been characterized as neural progenitors and shown to produce neurotrophic factors including brain derived neurotrophic factor (BDNF). To evaluate the therapeutic potential of ONPs on spinal cord injury, the rubrospinal pathway was selected because it has been shown to be BDNF dependent. Following partial cervical hemisection, an axotomized rubrospinal tract (RST) model of subacute spinal cord injury was used to determine if, 1) engrafted human adult NSFCs survived and integrated into the damaged spinal cord; 2) rescued axotomized rubrospinal neurons (RSN) from retrograde atrophy; 3) promoted axotomized RST axonal regeneration, reinnervation and forelimb functional recovery. Our long-term goal is to develop procedures through which a victim of spinal cord injury or other neurological degenerative conditions could serve as the donor of progenitors for his/her own restorative grafts without the need for immunosuppression or ethical controversy.Characterization of GFP-labeled human adult ONPs in vitroThe two randomly selected NSFC lines were transfected with green fluorescent protein (GFP) using the LERS-EGFP vector; approximately 60% of viable cells 4 days after transfection were fluorescent. In Minimal Essential Meduium (MEM) plus 2.5% fetal bovine serum (FBS), the GFP-labeled NSFCs (GFP-NSFCs) consisted of heterogeneous populations in which some cells exhibited immunoreactivity to nestin (~40%), neural cell adhesion molecule (NCAM, ~30%), β-tubulin Ⅲ (NST, ~95%), A2B5 (~5%), Trk pan (~20%), Trk A (~7%) and Trk B (~13%). No immunoreactivity wasobserved for glial fibrillary acidic protein (GFAP) and Trk C. Immunoreactivity for BDNF was detected throughout the cytoplasm and on the surface of most NSFCs (~ 90%).Consistently, the expression of mRNA and protein for BDNF in the two GFP-labeled NSFCs lines was demonstrated by RT-PCR and Western Blot respectively. By RT-PCR, cDNA amplification products representative of BDNF (298 bp) mRNA were detectable in RNA prepared from the two NSFCs lines. The immunoreactive bands for BDNF (14 kDa) were also observed in lysates of both NSFCs lines.To determine the endogenous and secreted levels of BDNF in the two GFP-NSFCs, cell culture supernates and cell lysates ELISA were performed. The concentration of BDNF in supernates was equivalent for both NSFCs, was 220.28 ± 8.42, 224.51 ± 7.91 pg/106 cells/ 24 h respectively. The amount of BDNF in lysates of both NSFCs lines was 39.28 ±7.51, 38.57 ± 8.43 pg/mg protein respectively.Furthermore, the bioactivity of cell culture supernates from the two NSFCs was assayed based on neurite outgrowth from embryonic chick DRG neurons. E8 chick DRG explants grown for 48 h in purified BDNF or supernates from GFP-labeled NSFCs showed extensive process outgrowth.In summary, differentiation potential of the two GFP- NSFCs in the MEM plus 2.5% FBS was restricted along a neuronal lineage ha... |
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