Distinct Glycosylation Responses to Spinal Cord Injury in Regenerative and Nonregenerative Models.

Traumatic spinal cord injury (SCI) results in disruption of tissue integrity and loss of function. We hypothesize that glycosylation has a role in determining the occurrence of regeneration and that biomaterial treatment can influence this glycosylation response. We investigated the glycosylation response to spinal cord transection in Xenopus laevis and rat. Transected rats received an aligned collagen hydrogel. The response compared regenerative success, regenerative failure, and treatment in an established nonregenerative mammalian system. In a healthy rat spinal cord, ultraperformance liquid chromatography (UPLC) N-glycoprofiling identified complex, hybrid, and oligomannose N-glycans. Following rat SCI, complex and outer-arm fucosylated glycans decreased while oligomannose and hybrid structures increased. Sialic acid was associated with microglia/macrophages following SCI. Treatment with aligned collagen hydrogel had a minimal effect on the glycosylation response. In Xenopus, lectin histochemistry revealed increased levels of N-acetyl-glucosamine (GlcNAc) in premetamorphic animals. The addition of GlcNAc is required for processing complex-type glycans and is a necessary foundation for additional branching. A large increase in sialic acid was observed in nonregenerative animals. This work suggests that glycosylation may influence regenerative success. In particular, loss of complex glycans in rat spinal cord may contribute to regeneration failure. Targeting the glycosylation response may be a promising strategy for future therapies.

Cell viability in three ex vivo rat models of spinal cord injury.

Spinal cord injury (SCI) is a devastating disorder that has a poor prognosis of recovery. Animal models of SCI are useful to understand the pathophysiology of SCI and the potential use of therapeutic strategies for human SCI. Ex vivo models of central nervous system (CNS) trauma, particularly mechanical trauma, have become important tools to complement in vivo models of injury in order to reproduce the sequelae of human CNS injury. Ex vivo organotypic slice cultures (OSCs) provide a reliable model platform for the study of cell dynamics and therapeutic intervention following SCI. In addition, these ex vivo models support the 3R concept of animal use in SCI research - replacement, reduction and refinement. Ex vivo models cannot be used to monitor functional recovery, nor do they have the intact blood supply of the in vivo model systems. However, the ex vivo models appear to reproduce many of the post traumatic events including acute and secondary injury mechanisms. Several well-established OSC models have been developed over the past few years for experimental spinal injuries ex vivo in order to understand the biological response to injury. In this study, we investigated cell viability in three ex vivo OSC models of SCI: stab injury, transection injury and contusion injury. Injury was inflicted in postnatal day 4 rat spinal cord slices. Stab injury was performed using a needle on transverse slices of spinal cord. Transection injury was performed on longitudinal slices of spinal cord using a double blade technique. Contusion injury was performed on longitudinal slices of spinal cord using an Infinite Horizon impactor device. At days 3 and 10 post-injury, viability was measured using dual staining for propidium iodide and fluorescein diacetate. In all ex vivo SCI models, the slices showed more live cells than dead cells over 10 days in culture, with higher cell viability in control slices compared with injured slices. Although no change in cell viability was observed between time-points in stab- and contusion-injured OSCs, a reduction in cell viability was observed over time in transection-injured OSCs. Taken together, ex vivo SCI models are a useful and reliable research tool that reduces the cost and time involved in carrying out animal studies. The use of OSC models provides a simple way to study the cellular consequences following SCI, and they can also be used to investigate potential therapeutics regimes for the treatment of SCI.

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments.

Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining, and by day 8 in the case of WFA. This study demonstrated neuronal cell surface glycosylation changes in an inhibitory environment and indicated a return to normal glycosylation after treatment with ChABC, which may be promising for identifying potential therapies for neuronal regeneration strategies.

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord.

BACKGROUND AIMS: In this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI). METHODS: Short hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. RESULTS: Animals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment. CONCLUSIONS: The improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.

Lectin Histochemistry for Tissues and Cells, and Dual Lectin and Antibody Co-localization.

In this chapter we describe in detail methods for lectin staining of (1) tissues, and (2) cells to identify and map endogenous glycosylation. We also describe (3) dual antibody and lectin staining of tissues to associate glycosylation with particular proteins or cells in tissues.

Thermosensitive hydrogel for prolonged delivery of lentiviral vector expressing neurotrophin-3 in vitro.

BACKGROUND: The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/ß-glycerol phosphate (ß-GP) as a carrier. METHODS: Three chitosan/ß-GP scaffolds were prepared with varying concentrations of chitosan and ß-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/ß-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre-seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth. RESULTS: Collagen displayed its most significant elution of virus on day 1 and chitosan/ß-GP (with a final concentration of 2.17% chitosan) on day 3. CONCLUSIONS: The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system.

Lentiviral Vectors Delivered with Biomaterials as Therapeutics for Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating trauma that can cause permanent disability, life-long chronic issues for sufferers and is a big socioeconomic burden. Regenerative medicine aims to overcome injury caused deficits and restore function after SCI through gene therapy and tissue engineering approaches. SCI has a multifaceted pathophysiology. Due to this, producing therapies that target multiple different cellular and molecular mechanisms might prove to be a superior approach in attempts at regeneration. Both biomaterials and nucleic acid delivery via lentiviral vectors (LVs) have proven to promote repair and restoration of function post SCI in animal models. Studies indicate that a combination of biomaterials and LVs is more effective than either approach alone. This review presents studies supporting the use of LVs and LVs delivered with biomaterials in therapies for SCI and summarises methods to combine LVs with biomaterials for SCI treatment. By summarising this knowledge this review aims to demonstrate how LV delivery with biomaterials can augment/compliment both LV and biomaterial therapeutic effects in SCI.

Lentiviral vector-mediated knockdown of the NG2 [corrected] proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar.

BACKGROUND: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). NG2, [corrected] one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. METHODS: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury. RESULTS AND DISCUSSION: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).

Engraftment, migration and differentiation of neural stem cells in the rat spinal cord following contusion injury.

BACKGROUND AIMS: Spinal cord injury is a devastating injury that impacts drastically on the victim's quality of life. Stem cells have been proposed as a therapeutic strategy. Neural stem (NS) cells have been harvested from embryonic mouse forebrain and cultured as adherent cells. These NS cells express markers of neurogenic radial glia. METHODS: Mouse NS cells expressing green fluorescent protein (GFP) were transplanted into immunosupressed rat spinal cords following moderate contusion injury at T9. Animals were left for 2 and 6 weeks then spinal cords were fixed, cryosectioned and analyzed. Stereologic methods were used to estimate the volume and cellular environment of the lesions. Engraftment, migration and differentiation of NS cells were also examined. RESULTS: NS cells integrated well into host tissue and appeared to migrate toward the lesion site. They expressed markers of neurons, astrocytes and oligodendrocytes at 2 weeks post-transplantation and markers of neurons and astrocytes at the 6-week time-point. NS cells appeared to have a similar morphologic phenotype to radial glia, in particular at the pial surface. CONCLUSIONS: Although no functional recovery was observed using the Basso Beattie Bresnahan (BBB) locomotor rating scale, NS cells are a potential cellular therapy for treatment of injured spinal cord. They may be used as delivery vehicles for therapeutic proteins because they show an ability to migrate toward the site of a lesion. They may also be used to replace lost or damaged neurons and oligodendrocytes.

Effect of cyclosporin A on functional recovery in the spinal cord following contusion injury.

Considerable evidence has shown that the immunosuppressant drug cyclosporin A (CsA) may have neuroprotective properties which can be exploited in the treatment of spinal cord injury. The aim of this study was to investigate the cellular environment within the spinal cord following injury and determine whether CsA has an effect on altering cellular interactions to promote a growth-permissive environment. CsA was administered to a group of rats 4 days after they endured a moderate contusion injury. Functional recovery was assessed using the Basso Beattie Bresnahan (BBB) locomotor rating scale at 3, 5 and 7 weeks post-injury. The rats were sacrificed 3 and 7 weeks post-injury and the spinal cords were sectioned, stained using histological and immunohistochemical methods and analysed. Using stereology, the lesion size and cellular environment in the CsA-treated and control groups was examined. Little difference in lesion volume was observed between the two groups. An improvement in functional recovery was observed within CsA-treated animals at 3 weeks. Although we did not see significant reduction in tissue damage, there were some notable differences in the proportion of individual cells contributing to the lesion. CsA administration may be used as a technique to control the cell population of the lesion, making it more permissive to neuronal regeneration once the ideal environment for regeneration and the effects of CsA administration at different time points post-injury have been identified.

Differential Glycosylation Expression in Injured Rat Spinal Cord Treated with Immunosuppressive Drug Cyclosporin-A.

Glycosylation is ubiquitous throughout the central nervous system and altered following spinal cord injury (SCI). The glial scar that forms following SCI is composed of several chondroitin sulfate proteoglycans, which inhibit axonal regrowth. Cyclosporin-A (CsA), an immunosuppressive therapeutic, has been proposed as a potential treatment after SCI. We investigated CsA treatment in the spinal cord of healthy, contusion injured, and injured CsA-treated rats. Lectin histochemistry using fluorescently labeled lectins, SBA, MAA, SNA-I, and WFA, was performed to identify the terminal carbohydrate residues of glycoconjugates within the spinal cord. SBA staining decreased in gray and white matter following spinal cord injury, whereas staining was increased at the lesion site in CsA-treated animals, indicating an increase in galactose and N-acetylgalactosamine terminal structures. No significant changes in MAA were observed. WFA staining was abundant in gray matter and observed to increase at the lesion site, in agreement with increased expression of chondroitin sulfate proteoglycans. SNA-I-stained blood vessels in all spinal cord regions and dual staining identified a subpopulation of astrocytes in the lesion site, which expressed α-(2,6)-sialic acid. Glycosylation were altered in injured spinal cord treated with CsA, indicating that glycosylation and alteration of particular carbohydrate structures are important factors to consider in the examination of the environment of the spinal cord after injury.

GDNF Schwann cells in hydrogel scaffolds promote regional axon regeneration, remyelination and functional improvement after spinal cord transection in rats.

Positively-charged oligo[poly(ethylene glycol)fumarate] (OPF+ ) is a biodegradable hydrogel used for spinal cord injury repair. We compared scaffolds containing primary Schwann cells (SCs) to scaffolds delivering SCs genetically modified to secrete high concentrations of glial cell-derived neurotrophic factor (GDNF). Multichannel OPF+ scaffolds loaded with SCs or GDNF-SCs were implanted into transected rat spinal cords for 4 weeks. GDNF-SCs promoted regeneration of more axons into OPF+ scaffolds (2773.0 ± 396.0) than primary SC OPF+ scaffolds (1666.0 ± 352.2) (p = 0.0491). This increase was most significant in central and ventral-midline channels of the scaffold. Axonal remyelination was quantitated by stereologic analysis. Increased myelination of regenerating axons was observed in the GDNF-SC group. Myelinating cell and axon complexes were formed by host SCs and not by implanted cells or host oligodendrocytes. Fast Blue retrograde tracing studies determined the rostral-caudal directionality of axonal growth. The number of neurons that projected axons rostrally through the GDNF-SC scaffolds was higher (7929 ± 1670) than in animals with SC OPF+ scaffolds (1069 ± 241.5) (p < 0.0001). The majority of ascending axons were derived from neurons located more than 15 mm from the scaffold-cord interface, and were identified to be lumbosacral intraspinal motor neurons. Transected animals with GDNF-SC OPF+ scaffolds partially recovered locomotor function at weeks 3 and 4 following surgery. Copyright © 2017 John Wiley & Sons, Ltd.

Therapeutic Effect of Neurotrophin-3 Treatment in an Injectable Collagen Scaffold Following Rat Spinal Cord Hemisection Injury.

Spinal cord injury (SCI) patients display varying quantities of spinal cord tissue damage with injuries that present as complete, incomplete or compressive. One theory proposed to repair the injured spinal cord and regain motor control is to regenerate axons through the lesion site. This study was designed to quantify the impact of a local injectable in situ forming hydrogel reservoir therapy following rat hemisection SCI. We investigated the effect of hydrogel only treatment following SCI in addition to hydrogels loaded with a neurotrophic factor, Neurotrophin-3 (NT-3), immediately following SCI. Functional recovery, assessed by Basso Beattie Bresnahan (BBB) locomotor test, and local healing mechanisms, including neuronal growth, glial scar formation, inflammation and collagen deposition were investigated one and 6 weeks postsurgery. Delivery of an injectable hydrogel significantly increased functional recovery at four and 6 weeks post injury. In addition, a significant reduction in the inhibitory glial scar and in inflammation was observed at the injury site. Similarly hydrogel + NT-3 delivered directly into the injury site significantly reduced glial scarring and collagen deposition. The hydrogel + NT-3 also resulted in a significant increase in neurons at 6 weeks post injury. This study represents a novel and effective therapy combining growth factor and a biomaterial based therapy following SCI.

A comparison of cell transplantation and retroviral gene transfection as tools to study lineage and differentiation in the rat spinal cord.

Establishing the cell lineage relationships of cells during development allows insight into when and where developmental decisions are made. In the developing spinal cord, the origin and fate of radial glial has yet to be determined. One way in which to address this question is to transplant enriched populations of radial glia into the ventricular zone (VZ) region of host embryos to examine the lineage and differentiation pattern of these cells. An indirect selection procedure using immunomagnetic beads (Dynabeads; Dynal Biotech) was used here to isolate spinal cord radial glia. This negative immunoselection procedure resulted in a high yield of radial glia. A fluorescent cytoplasmic dye (Cell Tracker Green CMFDA) was used to label radial glia before transplantation. The role of radial glia as progenitor cells can also be examined using a green fluorescent protein (GFP)-expressing retroviral vector. The retroviral vector allows dividing cells in the VZ region of the spinal cord to be tracked by labelling them with GFP. Both techniques were utilised here to successfully label and examine embryonic spinal cord radial glia in vivo after a microinjection of either fluorescently labelled radial glia or replication-incompetent GFP-expressing retrovirus in utero.

Robustness of MEK-ERK Dynamics and Origins of Cell-to-Cell Variability in MAPK Signaling.

Cellular signaling processes can exhibit pronounced cell-to-cell variability in genetically identical cells. This affects how individual cells respond differentially to the same environmental stimulus. However, the origins of cell-to-cell variability in cellular signaling systems remain poorly understood. Here, we measure the dynamics of phosphorylated MEK and ERK across cell populations and quantify the levels of population heterogeneity over time using high-throughput image cytometry. We use a statistical modeling framework to show that extrinsic noise, particularly that from upstream MEK, is the dominant factor causing cell-to-cell variability in ERK phosphorylation, rather than stochasticity in the phosphorylation/dephosphorylation of ERK. We furthermore show that without extrinsic noise in the core module, variable (including noisy) signals would be faithfully reproduced downstream, but the within-module extrinsic variability distorts these signals and leads to a drastic reduction in the mutual information between incoming signal and ERK activity.

Morphology and differentiation of radial glia in the developing rat spinal cord.

Important events underlying the proper functioning of the central nervous system (CNS) include the production, assembly, and differentiation of appropriate types and numbers of cells during development. The mechanisms that control these events are difficult to unravel because of displacement of cells from their sites of origin to their permanent locations and because of the diverse cellular composition of the CNS. As in other regions of the mammalian CNS, the two major classes of neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. In the developing spinal cord, radial glia are prominent. In this study, radial glia in the cervical region of the spinal cord were analysed. 1,1'Dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) was used to determine the morphology and distribution of radial glia during spinal cord development. The DiI labelling technique enabled locating glial precursor cells during spinal cord development. Radial fibres that extended from the central canal to the pial surface were present at embryonic days 14, 16, and 18 in the developing spinal cord. Their distribution was restricted with increasing development, and by embryonic day 20 the only remaining evidence of radial glia were short radial processes in the white matter.

Comparison of cellular architecture, axonal growth, and blood vessel formation through cell-loaded polymer scaffolds in the transected rat spinal cord.

The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF(+)) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF(+) scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF(+) scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.

Neurotrophic factor-expressing mesenchymal stem cells survive transplantation into the contused spinal cord without differentiating into neural cells.

The aim of this study was to assess the feasibility of transplanting mesenchymal stem cells (MSCs), genetically modified to express glial-derived neurotrophic factor (GDNF), to the contused rat spinal cord, and to subsequently assess their neural differentiation potential. MSCs expressing green fluorescent protein were transduced with a retroviral vector to express the neurotrophin GDNF. The transduction protocol was optimized by using green fluorescent protein-expressing retroviral constructs; approximately 90% of MSCs were transduced successfully after G418 selection. GDNF-transduced MSCs expressed the transgene and secreted growth factor into the media (approximately 12 ng/500,000 cells secreted into the supernatant 2 weeks after transduction). Injuries were established using an impactor device, which applied a given, reproducible force to the exposed spinal cord. GDNF-expressing MSCs were transplanted rostral and caudal to the site of injury. Spinal cord sections were analyzed 2 and 6 weeks after transplantation. We demonstrate that GDNF-transduced MSCs engraft, survive, and express the therapeutic gene up to 6 weeks posttransplantation, while maintaining an undifferentiated phenotype. In conclusion, transplanted MSCs have limited capacity for the replacement of neural cells lost as a result of a spinal cord trauma. However, they provide excellent opportunities for local delivery of neurotrophic factors into the injured tissue. This study underlines the therapeutic benefits associated with cell transplantation and provides a good example of the use of MSCs for gene delivery.

Gene-modified mesenchymal stem cells express functionally active nerve growth factor on an engineered poly lactic glycolic acid (PLGA) substrate.

Delivery of cellular and/or trophic factors to the site of injury may promote neural repair or regeneration and return of function after peripheral nerve or spinal cord injury. Engineered scaffolds provide a platform to deliver therapeutic cells and neurotrophic molecules. We have genetically engineered mesenchymal stem cells (MSCs) from the green rat (CZ-004 [SD TgN(act-EGFP)OsbCZ-004]) to express nerve growth factor (NGF) using an adenoviral vector. Cells maintained their stem cell phenotype as judged by expression of CD71 and CD172 markers, and absence of the hematopoietic marker CD45. Cells continued to express green fluorescent protein (GFP) on a long-term basis. Morphology, viability, and growth kinetics were maintained when cells were grown on a poly-lactic-co-glycolic acid (PLGA) polymer scaffold. Under appropriate growth conditions, they differentiated into chondrogenic, osteogenic, and adipogenic phenotypes, demonstrating that they retained their characteristics as MSCs. NGF was secreted from transduced MSCs at physiologically relevant levels ( approximately 25 ng/mL) measured by enzyme-linked immunoabsorbent assay (ELISA). Secreted NGF was functionally active in a neurite growth assay with PC12 cells. We conclude that MSCs are a good candidate for delivery of therapeutic factors into the injured nervous system. They are autologous, may be genetically modified to express neurotrophins, and are compatible with polymer surfaces that may be used as a potential delivery system.

Developmental potential of radial glia investigated by transplantation into the developing rat ventricular system in utero.

During development there is a clear correlation between position of dividing progenitor cells, mode of division and developmental potential, suggesting that the local environment of progenitor cells may influence their cell fate [ 17 (6), 639-647]. The contribution of these conditions was investigated here by transplantation of radial glial progenitor cells into isotopic, isochronic, heterotopic and heterochronic environment conditions. Neuronal cells were removed from E14 spinal cords using negative immunoselection. The remaining radial glia were transplanted into the ventricular system of host embryos and pups. Distance of migration as well as morphological and antigenic phenotype of transplanted radial glia was examined after various survival times post transplantation. Host age clearly influenced migration and differentiation of transplant cells, with transplant cells migrating further in younger hosts and differentiating earlier in older aged host environments. Evidence is presented showing that most transplanted spinal cord radial glia give rise to astrocytes. In addition some transplanted radial glia were shown to give rise to neurons in spinal cord regions. Radial glia did not appear to generate neurons in the brains of host animals until postnatal ages, perhaps because transplanted radial glia were isolated from spinal cord and thus may not have been influenced to behave as endogenous radial glia in the brain which commonly produce neurons.