Current clinical protocols use transient or continuous immunosuppression in patients receiving allogeneic neural precursor grafts for treatment of a variety of neurological diseases, including spinal trauma, stroke or ALS. At present there is no solid evidence, however, that would confirm long-term immune tolerance to allogeneic grafts in a large animal model(s) of chronic spinal cord injury. In our current study, we have tested the engraftment of porcine fetal NPCs after transplantation into the lumbar spinal cord of allogeneic transiently-immunosuppressed spinally-injured recipients. Material and Methods: Porcine fetal spinal cord-derived NPCs were isolated from 30-day-old fetuses. NPCs were expanded and characterized by immunofluorescence staining after differentiation in vitro. Clones of UBI-GFP expressing NPCs were then prepared and used in vivo for grafting into the lumbar spinal cord of allogeneic pigs with previous spinal cord L3 contusion injury. Animals (n=4) with fully developed paraplegia were grafted at 5.5 months after spinal injury. All animals received between 30-40 injections of NPCs targeted above, at epicenter and below the injury (10 ul/300,000 cells/injection). After cell grafting, animals were immunosuppressed iv with a combination of Prograf and MFF for 4 weeks. At 4 weeks, immunosuppression was terminated and animals survived for an additional 3 months (n=1), 6 months (n=1) or 12 months (n=2) without immunosuppression. After survival, animals were perfusion fixed and the presence and differentiation of grafted cells analyzed by immunofluorescence. Results: i) In vitro induced NPCs showed expression of neural/neuronal markers, including DCX, NeuN, GABA and GFAP. ii) Analysis of previously injured spinal cord tissue grafted with UBI-GFP+ NPCs showed extensive GFP+ grafts occupying previously injured spinal cord regions. iii) Double staining with neuronal markers (NeuN, SYN, NSE) showed the presence of high density grafted neurons throughout the grafts. iv) Staining with excitatory and inhibitory neurotransmitter markers showed a preferential GABA/glycin-ergic phenotype (VGAT and GAD65 +) in grafted neurons. v) Staining with glial markers showed near complete repopulation of individual grafts with GFAP+ astrocytes and regularly distributed Olig2 positive oligodendrocytes at 12 months after grafting. vi) No signs of cell-mediated rejection were seen at any time point after cell grafting. Conclusion: These data demonstrate that short course (4 weeks) immunosuppression is effective in inducing immune tolerance to allogeneic NPCs after grafting into chronically-injured spinal cord in pigs.
*M. MARSALA1, J. D. CIACCI2, E. I. CURTIS2, S. MARSALA1, M. R. NAVARRO1, P. CHEN1, S. JUHAS3, J. JUHASOVA3, K. YAMADA4, K. JOHE5;
1Dept. of Anesthesiol., 2Dept. of Neurosurg., Univ. of California San Diego, La Jolla, CA; 3Lab. of Cell Regeneration and Plasticity, Inst. of Animal Physiol. and Genet., Libechov, Czech Republic; 4Columbia Univ. Med. Ctr., New York, NY; 5Neuralstem, Germantown, MD
M. Marsala: None. J.D. Ciacci: None. E.I. Curtis: None. S. Marsala: None. M.R. Navarro: None. P. Chen: None. S. Juhas: None. J. Juhasova: None. K. Yamada: None. K. Johe: None.