Effects of Dibutyryl Cyclic-AMP on Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells Transplanted into Spinal Cord Injured Rats

Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (37±4%), followed by dbcAMP-microsphere treated NSPCs (27±14%) and untreated NSPCs (15±7%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels

A Preclinical Assessment of Neural Stem Cells as Delivery Vehicles for Anti-Amyloid Therapeutics

Transplantation of neural stems cells (NSCs) could be a useful means to deliver biologic therapeutics for late-stage Alzheimer's disease (AD). In this study, we conducted a small preclinical investigation of whether NSCs could be modified to express metalloproteinase 9 (MMP9), a secreted protease reported to degrade aggregated Aβ peptides that are the major constituents of the senile plaques. Our findings illuminated three issues with using NSCs as delivery vehicles for this particular application. First, transplanted NSCs generally failed to migrate to amyloid plaques, instead tending to colonize white matter tracts. Second, the final destination of these cells was highly influenced by how they were delivered. We found that our injection methods led to cells largely distributing to white matter tracts, which are anisotropic conduits for fluids that facilitate rapid distribution within the CNS. Third, with regard to MMP9 as a therapeutic to remove senile plaques, we observed high concentrations of endogenous metalloproteinases around amyloid plaques in the mouse models used for these preclinical tests with no evidence that the NSC-delivered enzymes elevated these activities or had any impact. Interestingly, MMP9-expressing NSCs formed substantially larger grafts. Overall, we observed long-term survival of NSCs in the brains of mice with high amyloid burden. Therefore, we conclude that such cells may have potential in therapeutic applications in AD but improved targeting of these cells to disease-specific lesions may be required to enhance efficacy

Long-Term Survival of Human Neural Stem Cells in the Ischemic Rat Brain upon Transient Immunosuppression

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders