Abstract
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.
Highlights
Repair after traumatic spinal cord injury (SCI) remains a significant challenge
neural stem/ progenitor cells (NSPCs) were cultured on chitosan films and exposed to varying concentrations of dibutyryl cyclic-AMP (dbcAMP) for 7 d, stained with the neuronal marker betaIII tubulin to quantify its effect on directed neuronal differentiation (Figure 1A)
By using the fully transected spinal cord injury model, we could clearly follow the effect of differentiated stem cells on tissue regeneration and locomotor functional recovery
Summary
Repair after traumatic spinal cord injury (SCI) remains a significant challenge. Extensive cell death and tissue disorganization, including demyelination, coupled with an inherent aborted and lack of spontaneous regeneration results in permanent disruption of signalling pathways. Resident endogenous neural stem cells exist lining the central canal of the adult spinal cord [1], their recruitment is limited in response to injury [2]. This is in large part due to the inhibitory environment of adult central nervous system (CNS) tissue, which is further exacerbated after injury. It is important to investigate the potential of neuronal replacement strategies for SCI as neurons are the core functional component of spinal cord signalling. Neuronal replacement after SCI is important to areas rich in neurons such as the cervical or thoraco-lumbar segments of the spinal cord
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