Implantation of adult bone marrow-derived mesenchymal stem cells transfected with the neurotrophin-3 gene and pretreated with retinoic acid in completely transected spinal cord.

Implantation of marrow-derived mesenchymal stem cells (MSCs) is the most promising therapeutic strategy for the treatment of spinal cord injury (SCI), especially because of their potential for clinical application, such as the avoidance of immunologic rejection, their strong secretory properties, and their plasticity for developing into neural cells. However, the recovery from SCI after MSC implantation is minimal due to their limited capacity for the reduction of cystic cavitation, for the axonal regeneration and their uncertain neural plasticity in the spinal cord. We previously pretreated MSCs with all-trans retinoic acid (RA) in vitro. Then we genetically modified them to overexpress neurotrophin-3 (NT-3) via a recombinant adenoviral vector (Adv). This combined treatment not only permitted more neuronal differentiation of MSCs, but stimulated more NT-3 secretion prior to grafting, according to our previous and present results. When these cells were implanted into the transected spinal cord of rats, the animals had some improvement (both functionally and structurally), including the recovery of hindlimb locomotor function, shown by the highest Basso, Beattie, and Bresnahan (BBB) scores, as well as dramatically reduced cavity volume, clear axonal regeneration and more neuronal survival. In contrast, simple MSC implantation is not a very effective therapy for spinal transection. However, the neuronal differentiation of MSCs after treatment with a combination of Adv-mediated NT-3 gene transfer and RA was only mildly improved in vivo.

Electro-acupuncture improves survival and migration of transplanted neural stem cells in injured spinal cord in rats.

This study investigated whether electro-acupuncture (EA) would improve the survival and migration of neural stem cells (NSCs) transplanted in injured spinal cord as well as the potential mechanisms. T10 spinal cord segments of 50 adult Sprague-Dawley (SD) rats were completely transected, and then NSCs were immediately transplanted into the transected site of the experimental animals, while control animals were sham operated without transplantation. Five days post-operation, electro-acupuncture treatment on GV9 (Zhiyang), GV6 (Jizhong), GV2 (Yaoshu) and GV1 (Changqiang) acupoints was applied for 14 days (EA+NSCs 14d) and 30 days (EA+NSCs 30d). ELISA and immunohistochemical staining were used to assess the content of neurotrophine-3 (NT-3) and the characteristics of transplanted NSCs. We found that the number of transplanted NSCs the survived in EA+NSCs14d group was significantly increased as compared to that of the NSCs30d group (5825.20 +/- 819.01 vs 4781.40 +/- 500.49, P<0.05). Immunostaining indicated that some transplanted NSCs developed into microtubule association protein 2 (MAP2) positive cells and many of them developed into glial fibrillary acidic protein (GFAP) positive cells in the NSCs30d group. Further, the migration length of transplanted NSCs toward caudal tissue in the injured site was longer in the EA+NSCs30d group than that in NSCs30d group (5.98 +/- 0.79 mm vs 3.96 +/- 1.72 mm; P<0.05). Also NT-3 in injured spinal cord tissue was 23% increased in the EA+NSCs14d group. These results suggest that the combination of EA and NSCs improves the survival and migration of NSCs in injured spinal cord in rats.

Recombinant adenovirus vector-mediated functional expression of neurotropin-3 receptor (TrkC) in neural stem cells.

We have constructed a recombinant adenovirus expression vector carrying the human neurotrophin-3 (NT-3) receptor TrkC (tyrosine protein kinase C) gene (rAd-TrkC; 2478 bp) and confirmed the expression of the encoded TrkC in green fluorescent protein (GFP)-murine neural stem cells (NSCs) by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunocytochemistry. The activity of the expressed rAd-TrkC was verified in vitro by evaluating dose-related responses of NSCs to NT-3, a TrkC specific ligand. TrkC-GFP-NSCs had a significantly higher percentage of neuronal differentiation when treated with NT-3 relative to the rAd-LacZ control cells (55.2% vs. 29.8%; P<0.05, chi(2) test). Thus, our rAd-TrkC vector can transfect NSCs and produce functional TrkC receptors to promote neuronal differentiation of NSCs.

Combination of adenoviral vector-mediated neurotrophin-3 gene transfer and retinoic acid promotes adult bone marrow cells to differentiate into neuronal phenotypes.

This study aims to investigate the effect of adenoviral vector-mediated neurotrophine-3 (NT-3) gene transfer and retinoic acid (RA) pretreatment on inducing neuronal differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro. MSCs could be efficiently transduced by NT-3 gene via recombinant adenoviral vectors (Adv). Combination of AdvNT-3 and RA significantly promoted MSCs to differentiate into cell types associated with phenotypes of neural lineages, which included neural markers nestin, NF, MAP2 and PSD95 as detected by immunocytochemistry. But the expressions of GFAP in these cells were not obvious. RT-PCR analysis revealed that AdvNT-3 in combination with RA pretreatment could initiate the transcription of TrkC mRNA. These results demonstrate that the combination of AdvNT-3 and RA pretreatment may promote neuronal differentiation of MSCs, which may serve as ideal seed cells for the repair of spinal cord injury.