Stem/progenitor cells derived from the ependymal region of the spinal cord have the ability to self-renew and are multipotential for neurons and glia. These cells may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. However, the optimal conditions for transplantation and the fate of transplanted cells are not fully known. In the current study, spinal cord stem/progenitor cells were cultured from adult male rats expressing enhanced green fluorescent protein (eGFP). Neurospheres were transplanted at the time of clip compression injury (35-g force) into the injury site, or 1 mm rostral and caudal to the injury site. Neurospheres were also transplanted into a subacute model (day 9 after injury) and a chronic model (day 28 after injury). Functional recovery was also studied in an acute injury model with weekly locomotor testing over a 16-week period. A significant increase in cell survival at 7 days was seen in rats receiving rostral and caudal injections as compared to injection directly into the site of injury. A significant increase in cell survival was also seen in rats receiving subacute transplants at 9 days after injury. Transplanted cells differentiated primarily into astrocytes (31.2%) and oligodendrocytes (50.3%), and a small number of neurons (1%). No improvement was seen in the Basso, Beattie and Bresnahan (BBB) locomotor rating scale after acute transplantation as compared with injury only, although surviving transplanted cells were identified that had migrated across the injury site from the rostral and caudal injection sites.

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Transplantation of neural stem and progenitor cells (NSPCs) is a promising strategy for repair after spinal cord injury. However, the epicenter of the severely damaged spinal cord is a hostile environment that results in poor survival of the transplanted NSPCs. We examined implantation of extramedullary chitosan channels seeded with NSPCs derived from transgenic green fluorescent protein (GFP) rats after spinal cord transection (SCT). At 14 weeks, we assessed the survival, maturation, and functional results using NSPCs harvested from the brain (brain group) or spinal cord (SC group) and seeded into chitosan channels implanted between the cord stumps after complete SCT. Control SCT animals had empty chitosan channels or no channels implanted. Channels seeded with brain or spinal cord-derived NSPCs showed a tissue bridge, although the bridges were thicker in the brain group. Both cell types showed long-term survival, but the number of surviving cells in the brain group was approximately five times as great as in the SC group. In both the brain and SC groups at 14 weeks after transplantation, many host axons were present in the center of the bridge in association with the transplanted cells. At 14 weeks astrocytic and oligodendrocytic differentiation in the channels was 24.8% and 17.3%, respectively, in the brain group, and 31.8% and 9.7%, respectively, in the SC group. The channels caused minimal tissue reaction in the adjacent spinal cord. There was no improvement in locomotor function. Thus, implantation of chitosan channels seeded with NSPCs after SCT created a tissue bridge containing many surviving transplanted cells and host axons, although there was no functional improvement.

BackgroundTraditionally, chronic wound infection is diagnosed by visual inspection under white light and microbiological sampling, which are subjective and suboptimal, respectively, thereby delaying diagnosis and treatment. To address this, we developed a novel handheld, fluorescence imaging device (PRODIGI) that enables non-contact, real-time, high-resolution visualization and differentiation of key pathogenic bacteria through their endogenous autofluorescence, as well as connective tissues in wounds.Methods and FindingsThis was a two-part Phase I, single center, non-randomized trial of chronic wound patients (male and female, ≥18 years; UHN REB #09-0015-A for part 1; UHN REB #12-5003 for part 2; clinicaltrials.gov Identifier: NCT01378728 for part 1 and NCT01651845 for part 2). Part 1 (28 patients; 54% diabetic foot ulcers, 46% non-diabetic wounds) established the feasibility of autofluorescence imaging to accurately guide wound sampling, validated against blinded, gold standard swab-based microbiology. Part 2 (12 patients; 83.3% diabetic foot ulcers, 16.7% non-diabetic wounds) established the feasibility of autofluorescence imaging to guide wound treatment and quantitatively assess treatment response. We showed that PRODIGI can be used to guide and improve microbiological sampling and debridement of wounds in situ, enabling diagnosis, treatment guidance and response assessment in patients with chronic wounds. PRODIGI is safe, easy to use and integrates into the clinical workflow. Clinically significant bacterial burden can be detected in seconds, quantitatively tracked over days-to-months and their biodistribution mapped within the wound bed, periphery, and other remote areas.ConclusionsPRODIGI represents a technological advancement in wound sampling and treatment guidance for clinical wound care at the point-of-care.Trial RegistrationClinicalTrials.gov NCT01651845; ClinicalTrials.gov NCT01378728

Neural stem/progenitor cells (NSPCs) capable of generating new neurons and glia reside in the adult mammalian spinal cord. Transplantation of NSPCs has therapeutic potential for spinal cord injury, although there is limited information on the ability of these cells to survive and differentiate in vivo. Neurospheres cultured from the periventricular region of the adult spinal cord contain NSPCs that are self-renewing and multipotent. We examined the survival, proliferation, migration, and differentiation of adult spinal cord NSPCs generated from green fluorescent protein (GFP) transgenic rats and transplanted into the intact spinal cord. The grafted GFP-expressing cells survived for at least 6 weeks in vivo and migrated from the injection site along the rostro-caudal axis of the spinal cord. Transplanted cells transiently proliferated following transplantation and approximately 17% of the GFP-positive cells were apoptotic at 1 day. Also, better survival was seen with NSPCs transplanted as neurospheres in comparison to NSPCs transplanted as dissociated cells. By 1 week posttransplantation, grafted cells primarily expressed an oligodendrocytic phenotype and only 2% differentiated into astrocytes. Approximately 75% versus 38% of the grafted cells differentiated into oligodendrocytes after transplantation into spinal white versus gray matter, respectively. This is the first report to examine the time course of cell survival, proliferation, apoptosis, and phenotypic differentiation of transplanted NSPSs in the spinal cord. This is also the first report to examine the differences between transplanted NSPCs grafted as neurospheres or dissociated cells, and to compare the differentiation potential after transplantation into spinal cord white versus gray matter.

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous beta tricalcium phosphate (beta-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.

Neural stem/progenitor cells capable of generating new neurons and glia, reside in specific areas of the adult mammalian central nervous system (CNS), including the ependymal region of the spinal cord and the subventricular zone (SVZ), hippocampus, and dentate gyrus of the brain. Much is known about the neurogenic regions in the CNS, and their response to various stimuli including injury, neurotrophins (NFs), morphogens, and environmental factors like learning, stress, and aging. This work has shaped our current views about the CNS's potential to recover lost tissue and function post-traumatically and the therapies to support the intrinsic regenerative capacity of the brain or spinal cord. Recently, intensive research has explored the potential of harvesting, culturing, and transplanting neural stem/progenitors as a therapeutic intervention for spinal cord injury (SCI) and traumatic brain injury (TBI). Another strategy has focused on maximizing the potential of this endogenous population of cells by stimulating their recruitment, proliferation, migration, and differentiation in vivo following traumatic lesions to the CNS. The promise of such experimental treatments has prompted tissue and biomaterial engineers to implant synthetic three-dimensional biodegradable scaffolds seeded with neural stem/progenitors into CNS lesions. Although there is no definitive answer about the ideal cell type for transplantation, strong evidence supports the use of region specific neural stem/progenitors. The technical and logistic considerations for transplanting neural stem/progenitors are extensive and crucial to optimizing and maintaining cell survival both before and after transplantation, as well as for tracking the fate of transplanted cells. These issues have been systematically addressed in many animal models, that has improved our understanding and approach to clinical therapeutic paradigms.