A rapid and sensitive method to measure numbers of live cells in alginate capsules following depolymerization with ethylenediaminetetraacetic acid.

Cell encapsulation in alginate prevents migration and extends cell survival in vivo while allowing the secretion of factors across semipermeable capsules. Confocal microscopy is used to measure numbers of cells/capsule, but is time-consuming and limited to capsule diameters <0.4 mm for accurate counting. A rapid, accurate and inexpensive method for measuring the number of cells per capsule by using 50 mM ethylenediaminetetraacetic acid to collapse capsules into a single plane was developed. This assay was used to accurately count the number of live cells/capsule for capsules crosslinked with 50 mM BaCl2 with diameters up to 0.8 mm. This assay is ideal for counting cells/capsule during optimization to scale up the production of encapsulated cells, and for determining dosing in translational studies.

Anti-Inflammatory Effects of Encapsulated Human Mesenchymal Stromal/Stem Cells and a Method to Scale-Up Cell Encapsulation.

Mesenchymal stem/stromal cells (MSC) promote recovery in a wide range of animal models of injury and disease. They can act in vivo by differentiating and integrating into tissues, secreting factors that promote cell growth and control inflammation, and interacting directly with host effector cells. We focus here on MSC secreted factors by encapsulating the cells in alginate microspheres, which restrict cells from migrating out while allowing diffusion of factors including cytokines across the capsules. One week after intrathecal lumbar injection of human bone marrow MSC encapsulated in alginate (eMSC), rat IL-10 expression was upregulated in distant rat spinal cord injury sites. Detection of human IL-10 protein in rostrally derived cerebrospinal fluid (CSF) indicated distribution of this human MSC-secreted cytokine throughout rat spinal cord CSF. Intraperitoneal (IP) injection of eMSC in a rat model for endotoxemia reduced serum levels of inflammatory cytokines within 5 h. Detection of human IL-6 in sera after injection of human eMSC indicates rapid systemic distribution of this human MSC-secreted cytokine. Despite proof of concept for eMSC in various disorders using animal models, translation of encapsulation technology has not been feasible primarily because methods for scale-up are not available. To scale-up production of eMSC, we developed a rapid, semi-continuous, capsule collection system coupled to an electrosprayer. This system can produce doses of encapsulated cells sufficient for use in clinical translation.

Efficacy of MSC in Patients with Severe COVID-19: Analysis of the Literature and a Case Study.

Patients with severe COVID-19 experience cytokine storm, an uncontrolled upregulation of pro-inflammatory cytokines, which if unresolved leads to acute respiratory distress syndrome (ARDS), organ damage, and death. Treatments with mesenchymal stromal cells (MSC) [Viswanathan S, Shi Y, Galipeau J, et al. Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy. 2019;21:1019-1024] appear to be effective in reducing morbidity and mortality. MSC respond to pro-inflammatory cytokines by releasing anti-inflammatory factors and mobilizing immune cells. We analyzed 82 COVID-19 clinical trials registered at ClinicalTrials.gov to determine MSC dosing, routes of administration, and outcome measures. Nearly all trials described the use of intravenous delivery with most doses ranging between 50 and 125 million MSC/treatment, which overlaps with a minimal effective dose range that we described previously. We also searched the literature to analyze clinical trial reports that used MSC to treat COVID-19. MSC were found to improve survival and oxygenation, increase discharge from intensive care units and hospitals, and reduce levels of pro-inflammatory markers. We report on a 91-year-old man with severe COVID-19 who responded rapidly to MSC treatment with transient reductions in several pro-inflammatory markers and delayed improvement in oxygenation. The results suggest that frequent monitoring of pro-inflammatory markers for severe COVID-19 will provide improved treatment guidelines by determining relationships between cytokine storms and ARDS. We propose that markers for cytokine storm are leading indicators for ARDS and that measurement of cytokines will indicate earlier treatment with MSC than is performed now for ARDS in severe COVID-19.

Trends in mesenchymal stem cell clinical trials 2004-2018: Is efficacy optimal in a narrow dose range?

The number of clinical trials using mesenchymal stem cells (MSCs) has increased since 2008, but this trend slowed in the past several years and dropped precipitously in 2018. Previous reports have analyzed MSC clinical trials by disease, phase, cell source, country of origin, and trial initiation date, all of which can be downloaded directly from ClinicalTrials.gov. We have extended analyses to a larger group of 914 MSC trials reported through 2018. To search for potential factors that may influence the design of new trials, we extracted data on routes of administration and dosing from individual ClinicalTrials.gov records as this information cannot be downloaded directly from the database. Intravenous (IV) injection is the most common, least invasive and most reproducible method, accounting for 43% of all trials. The median dose for IV delivery is 100 million MSCs/patient/dose. Analysis of all trials using IV injection that reported positive outcomes indicated minimal effective doses (MEDs) ranging from 70 to 190 million MSCs/patient/dose in 14/16 trials with the other two trials administering much higher doses of at least 900 million cells. Dose-response data showing differential efficacy for improved outcomes were reported in only four trials, which indicated a narrower MED range of 100-150 million MSCs/patient with lower and higher IV doses being less effective. The results suggest that it may be critical to determine MEDs in early trials before proceeding with large clinical trials.

Potential risk of clonally expanded amnion mesenchymal stem cell transplants in contused spinal cords.

OBJECTIVE: Mesenchymal stem/stromal cells (MSC) promote recovery after spinal cord injury (SCI) using adult bone marrow MSC (BM-MSC). Newborn tissues are a convenient source of MSC that does not involve an invasive procedure for cell collection. In this study the authors tested the effects of rat amnion MSC clone (rAM-MSC) in SCI. METHODS: We tested intra-parenchymal injection of a GFP+ rat rAM-MSC clone derived from E18.5 rats in rat SCI and measured behavioral recovery (BBB scores), histology and X-ray opacity. Expression of aggrecan was measured in culture after treatment with TGFß. RESULTS: Injection of rAM-MSC after SCI did not improve BBB scores compared to control vehicle injections; rather they reduced scores significantly over 6 weeks. Spinal cords injected with rAM-MSC were hard in regions surrounding the SCI site, which was confirmed by X-ray opacity. Whole mount imaging of these cords showed minimal tissue loss in the SCI site that occurred in SCI controls, and persistence of GFP+ rAM-MSC. Mason's Trichrome staining of tissue sections showed more intense staining for extracellular matrix (ECM) surrounding and extending beyond the SCI site with injections of rAM-MSC but not in controls. In response to TGF-ß treatment in culture, chondrogenic aggrecan was expressed at higher levels in rAM-MSC than in rBM-MSC, suggesting that the upregulation of TGF-ß in SCI sites may promote chondrogenic differentiation. CONCLUSION: Acute injection after SCI of a clonally expanded rAM-MSC resulted in aberrant differentiation towards a chondrocytic phenotype that disrupts the spinal cord and inhibits behavioral recovery after SCI. It will be critical to ensure that injection of extensively expanded neonatal cells do not differentiate aberrantly in traumatic CNS tissue and disrupt recovery.

Transcriptional Regulation of Notch1 Expression by Nkx6.1 in Neural Stem/Progenitor Cells during Ventral Spinal Cord Development.

Notch1 signaling plays a critical role in maintaining and determining neural stem/progenitor cell (NSPC) fate, yet the transcriptional mechanism controlling Notch1 specific expression in NSPCs remains incomplete. Here, we show transcription factor Nkx6.1 interacts with a cis-element (CR2, an evolutionarily conserved non-coding fragment in the second intron of Notch1 locus) and regulates the expression of Notch1 in ventral NSPCs of the developing spinal cord. We show that the Notch1 expression is modulated by the interaction of Nkx6.1 with a 139 bp enhancer sequence within CR2. Knockdown or overexpression of Nkx6.1 leads to down- or up-regulated Notch1 expression, respectively. In CR2-GFP transgenic mouse, GFP expression was found prominent in the ventricular zone and neural progenitor cells from embryonic day 9.5 to postnatal day 7. GFP+ cells were mainly neural progenitors for interneurons and not for motoneurons or glial cells. Moreover, GFP expression persisted in a subset of ependymal cells in the adult spinal cord, suggesting that CR2 is active in both embryonic and adult NSPCs. Together our data reveal a novel mechanism of Notch1 transcriptional regulation in the ventral spinal cord by Nkx6.1 via its binding with Notch1 enhancer CR2 during embryonic development.

Sizes and Sufficient Quantities of MSC Microspheres for Intrathecal Injection to Modulate Inflammation in Spinal Cord Injury.

Microencapsulation of mesenchymal stem cells (MSC) in alginate facilitates cell delivery, localization and survival, and modulates inflammation in vivo. However, we found that delivery of the widely used ~0.5 mm diameter encapsulated MSC (eMSC) by intrathecal injection into spinal cord injury (SCI) rats was highly variable. Injections of smaller (~0.2 mm) diameter eMSC into the lumbar spine were much more reproducible and they increased the anti-inflammatory macrophage response around the SCI site. We now report that injection of small eMSC >2 cm caudal from the rat SCI improved locomotion and myelin preservation 8 weeks after rat SCI versus control injections. Because preparation of sufficient quantities of small eMSC for larger studies was not feasible and injection of the large eMSC is problematic, we have developed a procedure to prepare medium-sized eMSC (~0.35 mm diameter) that can be delivered more reproducibly into the lumbar rat spine. The number of MSC incorporated/capsule in the medium sized capsules was ~5-fold greater than that in small capsules and the total yield of eMSC was ~20-fold higher than that for the small capsules. Assays with all three sizes of eMSC capsules showed that they inhibited TNF-α secretion from activated macrophages in co-cultures, suggesting no major difference in their anti-inflammatory activity in vitro. The in vivo activity of the medium-sized eMSC was tested after injecting them into the lumbar spine 1 day after SCI. Histological analyses 1 week later showed that eMSC reduced levels of activated macrophages measured by IB4 staining and increased white matter sparing in similar regions adjacent to the SCI site. The combined results indicate that ~0.35 mm diameter eMSC reduced macrophage inflammation in regions where white matter was preserved during critical early phases after SCI. These techniques enable preparation of eMSC in sufficient quantities to perform pre-clinical SCI studies with much larger numbers of subjects that will provide functional analyses of several critical parameters in rodent models for CNS inflammatory injury.

Long-term maintenance of Na+ channels at nodes of Ranvier depends on glial contact mediated by gliomedin and NrCAM.

Clustering of Na(+) channels at the nodes of Ranvier is coordinated by myelinating glia. In the peripheral nervous system, axoglial contact at the nodes is mediated by the binding of gliomedin and glial NrCAM to axonal neurofascin 186 (NF186). This interaction is crucial for the initial clustering of Na(+) channels at heminodes. As a result, it is not clear whether continued axon-glial contact at nodes of Ranvier is required to maintain these channels at the nodal axolemma. Here, we report that, in contrast to mice that lack either gliomedin or NrCAM, absence of both molecules (and hence the glial clustering signal) resulted in a gradual loss of Na(+) channels and other axonal components from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na(+) channels and ankyrin G, and then ßIV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na(+) channel clustering during development, but also contributes to the long-term maintenance of Na(+) channels at nodes of Ranvier.

NrCAM-regulating neural systems and addiction-related behaviors.

We have previously shown that a haplotype associated with decreased NrCAM expression in brain is protective against addiction vulnerability for polysubstance abuse in humans and that Nrcam knockout mice do not develop conditioned place preferences for morphine, cocaine or amphetamine. In order to gain insight into NrCAM involvement in addiction vulnerability, which may involve specific neural circuits underlying behavioral characteristics relevant to addiction, we evaluated several behavioral phenotypes in Nrcam knockout mice. Consistent with a potential general reduction in motivational function, Nrcam knockout mice demonstrated less curiosity for novel objects and for an unfamiliar conspecific, showed also less anxiety in the zero maze. Nrcam heterozygote knockout mice reduced alcohol preference and buried fewer marbles in home cage. These observations provide further support for a role of NrCAM in substance abuse including alcoholism vulnerability, possibly through its effects on behavioral traits that may affect addiction vulnerability, including novelty seeking, obsessive compulsion and responses to aversive or anxiety-provoking stimuli. Additionally, in order to prove glutamate homeostasis hypothesis of addiction, we analyzed glutamatergic molecules regulated by NRCAM expression. Glutaminase appears to be involved in NrCAM-related molecular pathway in two different tissues from human and mouse. An inhibitor of the enzyme, prolyl-leucyl-glycinamide, treatment produced, at least, some of the phenotypes of mice shown in alcohol preference and in anxiety-like behavior. Thus, NrCAM could affect addiction-related behaviors via at least partially modulation of some glutamatergic pathways and neural function in brain.

Nitration of Hsp90 induces cell death.

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

A cis-element in the Notch1 locus is involved in the regulation of gene expression in interneuron progenitors.

Interneurons comprise approximately one third of the total cortical neurons in the mammalian cerebral cortex. Studies have revealed many details in the generation of this cell type. However, the mechanism that defines interneuron-lineage specific gene expression is not well understood. Gene regulatory elements, e.g., promoters, enhancers, and trans-acting factors, are essential for the proper control of gene expression. Here, we report that a novel evolutionarily conserved cis-element in the second intron of the Notch1 locus plays an important role in regulating gene expression in interneuron progenitors. The spatiotemporal activity of the cis-element in the developing central nervous system (CNS) was determined by both transient reporter expression in the developing chick and a transgenic mouse model. Its activity is well correlated with neurogenesis in both the chick and mouse and restricted to neural progenitor cells in the ganglionic eminence that are fated to differentiate into GABAergic interneurons of the neocortex. We further demonstrate that the cis-element activity requires the binding motif for trans-acting factors Gsh1/Barx2/Brn3. Deletion of this binding motif abolishes reporter gene expression. Together, these data provide new insights into the regulatory mechanisms of interneuron development in the vertebrate CNS.

Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.

Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture.

Gamma-aminobutyric acid (GABA) ergic interneurons are lost in conditions including epilepsy and central nervous system injury, but there are few culture models available to study their function. Toward the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP(+) rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2, they exhibit varying patterns of differentiation. Transcriptional profiling and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5, and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In cocultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express ßIII-tubulin, and staining for synaptophysin and vesicular GABA transporter were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses.

E-cadherin-expressing feeder cells promote neural lineage restriction of human embryonic stem cells.

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article, we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule, E-cadherin, to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin, but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase, suggesting that E-cadherin promotes rapid neuronal differentiation. Further, these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin, neurofilament, and neural cell adhesion molecule. Moreover, blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation, we are able to utilize a specific cell-cell adhesion molecule, E-cadherin, to influence the nature and degree of neural specialization.

Encapsulated mesenchymal stromal cells for in vivo transplantation.

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.

Delayed intrathecal delivery of RhoA siRNA to the contused spinal cord inhibits allodynia, preserves white matter, and increases serotonergic fiber growth.

RhoA is a key regulator of the actin cytoskeleton that is upregulated after spinal cord injury (SCI). We analyzed different methods for siRNA delivery and developed siRNAs targeting RhoA (siRhoA) for SCI treatment. Cy 3.5-labeled siRNA delivered at the time of SCI yielded fluorescence in several cell types in the injury site. Intraspinal injections of chemically stabilized siRhoA into the spinal cord of injured rats reduced RhoA protein levels after 1 week and improved hindlimb walking over 6 weeks. To explore a less invasive route, we tested intrathecal injection of Cy 3.5-labeled siRNA via lumbar puncture 1 day after SCI, which resulted in robust uptake in the T9-T10 injury site. Lumbar injection of siRhoA 1 day after SCI reduced RhoA mRNA and protein levels 3 days after injection. Although siRhoA treatment did not yield significant improvement in locomotion, it decreased tactile hypersensitivity significantly compared to controls. Histological analysis at 8 weeks showed significant improvement in white matter sparing with siRhoA compared to control siRNA. siRhoA treatment also resulted in less accumulation of ED1+macrophages, increased PKC-γ immunoreactivity in the corticospinal tract rostral to the injury site, and increased serotonergic fiber growth 12 mm caudal to the contusion site. The ability of siRhoA to preserve white matter and promote serotonergic axonal regrowth caudal to the injury site is likely to suppress allodynia. This provides justification for considering clinical development of RhoA inhibitors to treat SCI sub-acutely to reduce allodynia, which occurs frequently in SCI patients.

Microscale plasma-initiated patterning of electrospun polymer scaffolds.

Microscale plasma-initiated patterning (µPIP) is a novel micropatterning technique used to create biomolecular micropatterns on polymer surfaces. The patterning method uses a polydimethylsiloxane (PDMS) stamp to selectively protect regions of an underlying substrate from oxygen plasma treatment resulting in hydrophobic and hydrophilic regions. Preferential adsorption of the biomolecules onto either the plasma-exposed (hydrophilic) or plasma-protected (hydrophobic) regions leads to the biomolecular micropatterns. In the current work, laminin-1 was applied to an electrospun polyamide nanofibrillar matrix following plasma treatment. Radial glial clones (neural precursors) selectively adhered to these patterned matrices following the contours of proteins on the surface. This work demonstrates that textured surfaces, such as nanofibrillar scaffolds, can be micropatterned to provide external chemical cues for cellular organization.

NrCAM deletion causes topographic mistargeting of thalamocortical axons to the visual cortex and disrupts visual acuity.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

F3/contactin and TAG1 play antagonistic roles in the regulation of sonic hedgehog-induced cerebellar granule neuron progenitor proliferation.

Modulation of the sonic hedgehog (SHH) pathway is a crucial factor in cerebellar morphogenesis. Stimulation of granule neuron progenitor (GNP) proliferation is a central function of SHH signalling, but how this is controlled locally is not understood. We show that two sequentially expressed members of the contactin (CNTN) family of adhesion molecules, TAG1 and F3, act antagonistically to control SHH-induced proliferation: F3 suppresses SHH-induced GNP proliferation and induces differentiation, whereas TAG1 antagonises F3. Production of GNPs in TAG1-null mice is delayed and reduced. F3 and TAG1 colocalise on GNPs with the related L1-like adhesion molecule NrCAM, and F3 fails to suppress the SHH-induced proliferation of NrCAM-deficient GNPs. We show that F3 and SHH both primarily affect a group of intermediate GNPs (IPs), which, though actively dividing, also express molecules associated with differentiation, including ß-tubulin III (TuJ1) and TAG1. In vivo, intermediate progenitors form a discrete layer in the middle of the external germinal layer (mEGL), while F3 becomes expressed on the axons of postmitotic granule neurons as they leave the inner EGL (iEGL). We propose, therefore, that F3 acts as a localised signal in the iEGL that induces SHH-stimulated cells in the overlying mEGL to exit cell cycle and differentiate. By contrast, expression of TAG1 on GNPs antagonises this signal in the mEGL, preventing premature differentiation and sustaining GNP expansion in a paracrine fashion. Together, these findings indicate that CNTN and L1-like proteins play a significant role in modulating SHH-induced neuronal precursor proliferation.

Juvenile and adult olfactory ensheathing cells bundle and myelinate dorsal root ganglion axons in culture.

Olfactory ensheathing cells (OEC), which normally associate closely with but do not myelinate axons in situ, myelinate axons in the adult mammalian spinal cord. They are of clinical interest as candidate cells for autologous transplantation but the ability of OEC to myelinate axons in vitro has been controversial. To clarify this issue, we isolated OEC from olfactory bulbs (OB) of juvenile and adult rats expressing GFP and analyzed their ability to myelinate axons. Using a well-defined assay for myelination of dorsal root ganglia (DRG) axons in culture, we found that OEC from juvenile pups associated with and then myelinated DRG axons. OEC assembled into bundles with the axons by 1week and required more than a week before myelination on axons was detected. In contrast, rat Schwann cells did not bundle axons and they formed P0(+) and MBP(+) myelin segments after as little as 1week. Most of the OEC in culture exhibited staining for calponin, a marker that was not found on Schwann cells in culture, whereas in both OEC and Schwann cell populations nearly all cells were positive for p75NTR and GFAP. These results confirm previous reports showing only subtle immunological differences between Schwann cells and OEC. Besides differences in the rate of myelination, we detected two additional functional differences in the interactions of OEC and Schwann cells with DRG axons. First, the diameter of OEC generated myelin was greater than for Schwann cell myelin on DRG axons. Second, OEC but not Schwann cells myelinated DRG axons in the absence of vitamin C. OEC isolated from adult OB were also found to bundle and myelinate DRG axons but the latter occurred only after incubation times of at least 3weeks. The results indicate that adult OEC require longer incubation times than juvenile OEC to myelinate axons and suggest that patterns of myelination by OEC and Schwann cells are distinguishable at least on axons in vitro. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.