An Alzheimer's Disease Patient-Derived Olfactory Stem Cell Model Identifies Gene Expression Changes Associated with Cognition.

An early symptom of Alzheimer's disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore changes of early AD-associated pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

Claudin-5 binder enhances focused ultrasound-mediated opening in an in vitro blood-brain barrier model.

Rationale: The blood-brain barrier (BBB) while functioning as a gatekeeper of the brain, impedes cerebral drug delivery. An emerging technology to overcome this limitation is focused ultrasound (FUS). When FUS interacts with intravenously injected microbubbles (FUS+MB), the BBB opens, transiently allowing the access of therapeutic agents into the brain. However, the ultrasound parameters need to be tightly tuned: when the acoustic pressure is too low there is no opening, and when it is too high, tissue damage can occur. We therefore asked whether barrier permeability can be increased by combining FUS+MB with a second modality such that in a clinical setting lower acoustic pressures could be used. Methods: Given that FUS+MB achieves BBB opening in part by disruption of tight junction (TJ) proteins such as claudin-5 of brain endothelial cells, we generated a stable MDCK (Madin-Darby Canine Kidney) II cell line (eGFP-hCldn5-MDCK II) that expresses fluorescently tagged human claudin-5. Two claudin-5 binders, the peptide mC5C2 and cCPEm (truncated form of an enterotoxin), reported previously to weaken the barrier, were synthesized and assessed for their abilities to enhance the permeability of cellular monolayers. We then performed a comparative analysis of single and combination treatments, measuring transendothelial electrical resistance (TEER) and cargo leakage, combined with confocal image analysis. Results: We successfully generated a novel cell line that formed functional monolayers as validated by an increased TEER reading and a low (< 0.2%) permeability to sodium fluorescein (376 Da). We found that the binders exerted a time- and concentration-dependent effect on barrier opening when incubated over an extended period, whereas FUS+MB caused a rapid opening followed by recovery after 12 hours within the tested pressure range. Importantly, preincubation with cCPEm prior to FUS+MB treatment resulted in greater barrier opening compared to either FUS+MB or cCPEm alone as measured by reduced TEER values and an increased permeability to fluorescently labelled 40 kDa dextran (FD40). Conclusion: The data suggest that pre incubation with clinically suitable binders to TJ proteins may be a general strategy to facilitate safer and more effective ultrasound-mediated BBB opening in cellular and animal systems and potentially also for the treatment of human diseases of the brain.

Oxidative Stress-Induced Axon Fragmentation Is a Consequence of Reduced Axonal Transport in Hereditary Spastic Paraplegia SPAST Patient Neurons.

Hereditary spastic paraplegia (HSP) is a group of inherited disorders characterized by progressive spasticity and paralysis of the lower limbs. Autosomal dominant mutations in SPAST gene account for ∼40% of adult-onset patients. We have previously shown that SPAST patient cells have reduced organelle transport and are therefore more sensitive to oxidative stress. To test whether these effects are present in neuronal cells, we first generated 11 induced pluripotent stem (iPS) cell lines from fibroblasts of three healthy controls and three HSP patients with different SPAST mutations. These cells were differentiated into FOXG1-positive forebrain neurons and then evaluated for multiple aspects of axonal transport and fragmentation. Patient neurons exhibited reduced levels of SPAST encoded spastin, as well as a range of axonal deficits, including reduced levels of stabilized microtubules, lower peroxisome transport speed as a consequence of reduced microtubule-dependent transport, reduced number of peroxisomes, and higher density of axon swellings. Patient axons fragmented significantly more than controls following hydrogen peroxide exposure, suggesting for the first time that the SPAST patient axons are more sensitive than controls to the deleterious effects of oxidative stress. Treatment of patient neurons with tubulin-binding drugs epothilone D and noscapine rescued axon peroxisome transport and protected them against axon fragmentation induced by oxidative stress, showing that SPAST patient axons are vulnerable to oxidative stress-induced degeneration as a consequence of reduced axonal transport.

Altered Brain Endothelial Cell Phenotype from a Familial Alzheimer Mutation and Its Potential Implications for Amyloid Clearance and Drug Delivery.

The blood-brain barrier (BBB) presents a barrier for circulating factors, but simultaneously challenges drug delivery. How the BBB is altered in Alzheimer disease (AD) is not fully understood. To facilitate this analysis, we derived brain endothelial cells (iBECs) from human induced pluripotent stem cells (hiPSCs) of several patients carrying the familial AD PSEN1 mutation. We demonstrate that, compared with isogenic PSEN1 corrected and control iBECs, AD-iBECs exhibit altered tight and adherens junction protein expression as well as efflux properties. Furthermore, by applying focused ultrasound (FUS) that transiently opens the BBB and achieves multiple therapeutic effects in AD mouse models, we found an altered permeability to 3-5 kDa dextran as a model cargo and the amyloid-ß (Aß) peptide in AD-iBECs compared with control iBECs. This presents human-derived in vitro models of the BBB as a valuable tool to understand its role and properties in a disease context, with possible implications for drug delivery.

A Patient-Specific Stem Cell Model to Investigate the Neurological Phenotype Observed in Ataxia-Telangiectasia.

The molecular pathogenesis of ataxia-telangiectasia (A-T) is not yet fully understood, and a versatile cellular model is required for in vitro studies. The occurrence of continuous neurogenesis and easy access make the multipotent adult stem cells from the olfactory mucosa within the nasal cavity a potential cellular model. We describe an efficient method to establish neuron-like cells from olfactory mucosa biopsies derived from A-T patients for the purpose of studying the cellular and molecular aspects of this debilitating disease.

Cell migration in schizophrenia: Patient-derived cells do not regulate motility in response to extracellular matrix.

Schizophrenia is a highly heritable psychiatric disorder linked to a large number of risk genes. The function of these genes in disease etiology is not fully understood but pathway analyses of genomic data suggest developmental dysregulation of cellular processes such as neuronal migration and axon guidance. Previous studies of patient-derived olfactory cells show them to be more motile than control-derived cells when grown on a fibronectin substrate, motility that is dependent on focal adhesion kinase signaling. The aim of this study was to investigate whether schizophrenia patient-derived cells are responsive to other extracellular matrix (ECM) proteins that bind integrin receptors. Olfactory neurosphere-derived cells from nine patients and nine matched controls were grown on ECM protein substrates at increasing concentrations and their movement was tracked for 24h using automated high-throughput imaging. Control-derived cells increased their motility as the ECM substrate concentration increased, whereas patient-derived cell motility was little affected by ECM proteins. Patient and control cells had appropriate integrin receptors for these ECM substrates and detected them as shown by increases in focal adhesion number and size in response to ECM proteins, which also induced changes in cell morphology and cytoskeleton. These observations indicate that patient cells failed to translate the detection of ECM proteins into appropriate changes in cell motility. In a sense, patient cells act like a moving car whose accelerator is jammed, moving at the same speed without regard to the external environment. This focuses attention on cell motility regulation rather than speed as key to impairment of neuronal migration in the developing brain in schizophrenia.

Schizophrenia patient-derived olfactory neurosphere-derived cells do not respond to extracellular reelin.

Reelin expression is reduced in various regions in the post-mortem brain of schizophrenia patients but the exact role of reelin function in the neurobiology of schizophrenia remains elusive. Absence of reelin in knockout mouse causes inverted lamination of the neocortex due to aberrant neuronal migration. The aim of this study was to utilize patient-derived olfactory neurosphere-derived (ONS) cells to investigate whether extracellular reelin alters cell motility in schizophrenia patient-derived cells. ONS cells from nine patients were compared with cells from nine matched healthy controls. Automated high-throughput imaging and analysis were used to track motility of individual living cells on reelin-coated surfaces produced from reelin secreted into the medium by HEK293FT cells transfected with the full-length reelin plasmid pCrl. Automated assays were used to quantify intracellular cytoskeleton composition, cell morphology, and focal adhesions. Expression of reelin and components of the reelin signaling pathway were measured by western blot and flow cytometry. Reelin inhibited the motility of control cells but not patient cells, and increased the number and size of focal adhesions in control cells but not patient cells. Patient and control cells expressed similar levels of the reelin receptors and the reelin signaling protein, Dab1, but patient cells expressed less reelin. Patient cells were smaller than control cells and had less actin and acetylated α-tubulin, components of the cytoskeleton. These findings are the first direct evidence that cellular responses to reelin are impaired in schizophrenia and are consistent with the role of reelin in cytoarchitectural deficits observed in schizophrenia patient brains.

Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress.

Rapid growth in early childhood associated with young adult overweight and obesity--evidence from a community based cohort study.

BACKGROUND: Rapid weight gain in early life may increase the risk of overweight and obesity in adulthood. We investigated the association between the rate of growth during early childhood and the development of overweight and obesity in young adults. METHODS: We used a prospective cohort study of 2077 young adults who were born between 1981 and 1984 in Brisbane, Australia and had anthropometry measurements available at birth, 6 months, 5 years, 14 years and 21 years of age. The associations of rate of early growth with body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) and their categories at 21 years were studied using multivariate analysis. RESULTS: We found that rapid weight gain [> + 0.67 standard deviation score (SDS)] in the first 5 years of life was associated with young adults' overweight status (BMI: adjusted OR = 2.35, 95% CI, 1.82-3.03; WC: adjusted OR = 2.20, 95% CI, 1.65-2.95). We also observed that slow weight gain in the first 5 years of age (< -0.67 SDS) was inversely associated with overweight (BMI: OR = 0.62, 95% CI, 0.45-0.84). Such associations were not found with WHR. Rapid weight gain in the first 6 months of life increased the risk of overweight as defined by BMI (adjusted OR = 1.13, 95% CI, 0.86-1.49) and WC (adjusted OR = 1.24, 95% CI, 0.92-1.67), but these associations were not statistically significant. CONCLUSION: Rapid weight gain in the first 5 years of life in children increased their risk of a higher BMI and WC in young adulthood, in contrast slow weight gain was inversely associated with weight status at 21 years.

Low dose tubulin-binding drugs rescue peroxisome trafficking deficit in patient-derived stem cells in Hereditary Spastic Paraplegia.

Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials.

Cesarean delivery and the long-term risk of offspring obesity.

OBJECTIVE: To examine the association between the mode of delivery and the risk of offspring obesity by age 21 years using a large community-based birth cohort study in Australia. METHODS: We followed-up a subsample of 2,625 offspring for whom we had measured physical assessments, including height and weight at 21 years and hospital-recorded mode of delivery, in the Mater Hospital in Brisbane, Australia, between 1981 and 1983. Body mass index (BMI) and waist circumference were measured at 21 years. Multivariable regression analysis was used to examine the independent associations of mode of delivery with offspring BMI and waist circumference. RESULTS: In the cohort, 12.1% were born by cesarean delivery. Maternal and birth factors independently associated with the mode of delivery were age, overweight and obesity status, smoking status during pregnancy, hypertensive disorder during pregnancy, and neonatal low birth weight. By 21 years, 21.5% of offspring were overweight and 12.4% were obese. Offspring overweight and obesity status, as well as BMI and waist circumference, were not associated with the mode of delivery. CONCLUSIONS: Findings of this study do not support the idea that cesarean delivery has increased the risk of long-term offspring obesity. LEVEL OF EVIDENCE: : III.

A patient-derived olfactory stem cell disease model for ataxia-telangiectasia.

The autosomal recessive disorder ataxia-telangiectasia (A-T) is characterized by genome instability, cancer predisposition and neurodegeneration. Although the role of ataxia-telangiectasia mutated (ATM) protein, the protein defective in this syndrome, is well described in the response to DNA damage, its role in protecting the nervous system is less clear. We describe the establishment and characterization of patient-specific stem cells that have the potential to address this shortcoming. Olfactory neurosphere (ONS)-derived cells were generated from A-T patients, which expressed stem cell markers and exhibited A-T molecular and cellular characteristics that included hypersensitivity to radiation, defective radiation-induced signaling and cell cycle checkpoint defects. Introduction of full-length ATM cDNA into these cells corrected defects in the A-T cellular phenotype. Gene expression profiling and pathway analysis revealed defects in multiple cell signaling pathways associated with ATM function, with cell cycle, cell death and DNA damage response pathways being the most significantly dysregulated. A-T ONS cells were also capable of differentiating into neural progenitors, but they were defective in neurite formation, number of neurites and length of these neurites. Thus, ONS cells are a patient-derived neural stem cell model that recapitulate the phenotype of A-T, do not require genetic reprogramming, have the capacity to differentiate into neurons and have potential to delineate the neurological defect in these patients.

A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations.

Hereditary spastic paraplegia (HSP) leads to progressive gait disturbances with lower limb muscle weakness and spasticity. Mutations in SPAST are a major cause of adult-onset, autosomal-dominant HSP. Spastin, the protein encoded by SPAST, is a microtubule-severing protein that is enriched in the distal axon of corticospinal motor neurons, which degenerate in HSP patients. Animal and cell models have identified functions of spastin and mutated spastin but these models lack the gene dosage, mutation variability and genetic background that characterize patients with the disease. In this study, this genetic variability is encompassed by comparing neural progenitor cells derived from biopsies of the olfactory mucosa from healthy controls with similar cells from HSP patients with SPAST mutations, in order to identify cell functions altered in HSP. Patient-derived cells were similar to control-derived cells in proliferation and multiple metabolic functions but had major dysregulation of gene expression, with 57% of all mRNA transcripts affected, including many associated with microtubule dynamics. Compared to control cells, patient-derived cells had 50% spastin, 50% acetylated α-tubulin and 150% stathmin, a microtubule-destabilizing enzyme. Patient-derived cells were smaller than control cells. They had altered intracellular distributions of peroxisomes and mitochondria and they had slower moving peroxisomes. These results suggest that patient-derived cells might compensate for reduced spastin, but their increased stathmin expression reduced stabilized microtubules and altered organelle trafficking. Sub-nanomolar concentrations of the microtubule-binding drugs, paclitaxel and vinblastine, increased acetylated α-tubulin levels in patient cells to control levels, indicating the utility of this cell model for screening other candidate compounds for drug therapies.

NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson's disease.

BACKGROUND: Without appropriate cellular models the etiology of idiopathic Parkinson's disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson's disease. RESULTS: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H(2)O(2) than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson's Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson's disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment. CONCLUSIONS: Our results confirmed NRF2 as a potential therapeutic target for Parkinson's disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.

Disease-specific, neurosphere-derived cells as models for brain disorders.

There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.

Selected changes in spinal cord morphology after T4 transection and olfactory ensheathing cell transplantation.

Spinal cord transection at T4 results in severe damage of the nervous tissue, with impairment of motor, sensory and autonomic functions. Transplantation of olfactory ensheathing cells (OECs) has the potential to improve these functions through a number of mechanisms, which include facilitation of regeneration and neuroprotection. For cardiovascular functions, we have previously shown that OECs reduce the duration of autonomic dysreflexia, without evidence of regeneration. To further understand the mechanisms underpinning this improvement, we have studied changes in selected morphological features (cavitation, non-cavity tissue loss, morphology of sympathetic preganglionic neurons and primary afferent fibre density) in the T4-transected rat spinal cord over 9 weeks, both in control and OEC-transplanted animals. T4 transection led to a number of structural changes: gradual formation of cavities, non-cavity tissue loss, a long-term increase in soma size of sympathetic preganglionic neurons and a temporary increase in the extent of their dendritic arbours, and an increase in the density of primary afferent fibres caudal to the lesion. OECs decreased the cavitation and normalised soma size of the sympathetic preganglionic neurons below the lesion, while increasing the extent of dendritic arbours in the preganglionic neurons above the lesion. Thus the OECs may contribute to the normalisation of the dysreflexic hypertension through tissue preservation and normalisation of the morphology of the preganglionic neurons caudal to the lesion, while enhancing the input on the rostral preganglionic neurons, whose vasomotor control remains intact. We hypothesise that these changes are mediated through secretion of soluble trophic factors by the transplanted OECs.

Effects of human OEC-derived cell transplants in rodent spinal cord contusion injury.

Numerous reports indicate that rodent olfactory ensheathing cells (OECs) assist in spinal cord repair and clinical trials have been undertaken using autologous transplantation of human olfactory ensheathing cells (hOECs) as a treatment for spinal cord injury. However, there are few studies investigating the efficacy of hOECs in animal models of spinal cord injury. In this study hOECs were derived from biopsies of human olfactory mucosa, purified by culture in a serum-free medium containing neurotrophin-3, genetically labelled with EGFP, and stored frozen. These hOEC-derived cells were thawed and transplanted into the spinal cord injury site 7 days after a moderate contusion injury of the spinal cord at thoracic level T10 in the athymic rat. Six weeks later the animals receiving the hOEC-derived transplants had greater functional improvement in their hindlimbs than controls, assessed using open field (BBB scale) and horizontal rung walking tests. Histological analysis demonstrated beneficial effects of hOEC-derived cell transplantation: reductions in the volume of the lesion and the cavities within the lesion. The transplanted cells were located at the periphery of the lesion where they integrated with GFAP-positive astrocytes resulting in a significant reduction of GFAP staining intensity adjacent to the lesion. Although their mechanism of action is unclear we conclude that hOEC-derived cell transplants improved functional recovery after transplantation into the contused spinal cord, probably by modulating inflammatory responses and reducing secondary damage to the cord.

Olfactory ensheathing cells reduce duration of autonomic dysreflexia in rats with high spinal cord injury.

Autonomic dysreflexia is a common complication in high spinal cord injury and can result in serious consequences and death. Here we have examined the effect of acute transplantation of olfactory ensheathing cells on cardiovascular functions in rats. After T4 transection, radio-telemetric recording in conscious animals was used to study blood pressure and heart rate at rest and during autonomic dysreflexia for up to 8 weeks post-injury. Olfactory ensheathing cells from syngeneic rats were transplanted at the injury site; control animals received culture medium only. At the study end point, we examined morphometric features of sympathetic preganglionic neurons above and below the injury. T4 transection resulted in a fall in resting mean arterial pressure and an increase in resting heart rate. Colorectal distension, used to trigger autonomic dysreflexia, caused episodic hypertension and bradycardia. Although the cell transplantation had no effect on resting cardiovascular parameters, it led to a significantly faster recovery from hypertension, with the recovery time shortened by approximately 25%. The transection resulted in an increase in soma size of sympathetic preganglionic neurons above and below the injury. OEC transplantation normalised this change below the injury and increased dendritic length of preganglionic neurons above the injury, compared to controls. It has been proposed that changes in sympathetic preganglionic neurons following spinal cord transection may be related to the development of autonomic dysreflexia. Our results suggest that olfactory ensheathing cells may alter the morphology of these neurons, and hence modify their activity in the neuronal networks responsible for the dysreflexic reaction.

A novel cell transplantation protocol and its application to an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, which selectively affects motor neurons throughout the central nervous system. The extensive distribution of motor neurons is an obstacle to applying cell transplantation therapy for the treatment of ALS. To overcome this problem, we developed a cell transplantation method via the fourth cerebral ventricle in mice. We used mouse olfactory ensheathing cells (OECs) and rat mesenchymal stem cells (MSCs) as donor cells. OECs are reported to promote regeneration and remyelination in the spinal cord, while MSCs have a capability to differentiate into several types of specific cells including neural cells. Furthermore both types of cells can be relatively easily obtained by biopsy in human. Initially, we confirmed the safety of the operative procedure and broad distribution of grafted cells in the spinal cord using wild-type mice. After transplantation, OECs distributed widely and survived as long as 100 days after transplantation, with a time-dependent depletion of cell number. In ALS model mice, OEC transplantation revealed no adverse effects but no significant differences in clinical evaluation were found between OEC-treated and non-transplanted animals. After MSC transplantation into the ALS model mice, females, but not males, showed a statistically longer disease duration than the non-transplanted controls. We conclude that intrathecal transplantation could be a promising way to deliver donor cells to the central nervous system. Further experiments to elucidate relevant conditions for optimal outcomes are required.