Comparison of Bulbar and Mucosal Olfactory Ensheathing Cells Using FACS and Simultaneous Antigenic Bivariate Cell Cycle Analysis

Transplantation of olfactory ensheathing cells (OECs) is a promising route for CNS repair. There have, however, been major discrepancies between the results from different groups. Part of this can be attributed to variations in cell sources and culture protocols. Accurate estimation of the proportions of OECs and their associated fibroblasts (ONFs) and their evolution with time in culture is an essential baseline for establishing the reparative properties of transplants. In this study, we compare the evolution of cultures from the superficial layers of the olfactory bulb with tissue from the olfactory mucosa, both whole and split into lamina propria and epithelial layer. We used FACS based on p75 and Thy1 to provide a robust and objective numerical estimate of the numbers of OECs and ONFs, respectively in the cultures. A novel four color simultaneous antigenic bivariate cell cycle analysis shows that proliferation of OECs is time-limited, and is unable to prevent an overall loss of OECs with time. Overall, the numbers of OECs in the cultures were inversely correlated with the deposition of fibronectin (FN). Further, culture of the cells purified by flow cytometry shows that, whereas the Thy1 population is terminally differentiated, the p75 population from the mucosal samples generates subpopulations with different antigenic phenotypes, including the reappearance of a subpopulation of p75 cells expressing FN. Culturing epithelial samples at high density reveals an unexpected transient stem cell-like population of rapidly proliferating p75 positive cells. (C) 2011 Wiley-Liss, Inc

Directionality and bipolarity of olfactory ensheathing cells on electrospun nanofibers

AIM: As a preliminary to the construction of olfactory ensheathing cells (OECs) bearing scaffold for bridging larger lesions in the spinal cord, we have investigated the response of purified cultured OECs to nanoscale fibers of varying diameter using US FDA-approved, biodegradable poly(lactic-co-glycolic-acid). MATERIALS & METHODS: Conventional electrospinning produced fibers of approximately 700 nm diameter (nano-700) while nanocomposite electrospinning with quantum dots produced significantly more uniform fibers of a reduced diameter to approximately 237 nm (nano-250). OECs from adult rat were FACS purified, cultured at low density on either a flat surface or a meshwork of randomly orientated nano-700 and nano-250 fibers, and assessed using cytomorphometric analysis of immunofluorescent confocal images and by scanning electron microscopy. RESULTS & CONCLUSION: Compared with a flat surface, culture on a nano-700 mesh increases cell attachment. Cells change from rounded to stellate forms in random orientation. Further size reduction to the nano-250 favors bipolarity in cells with unidirectional orientation as observed in the case when transplanted OECs were used to bridge areas of damage in rat spinal cords

Functional Regeneration of Supraspinal Connections in a Patient With Transected Spinal Cord Following Transplantation of Bulbar Olfactory Ensheathing Cells With Peripheral Nerve Bridging

Treatment of patients sustaining a complete spinal cord injury remains an unsolved clinical problem because of the lack of spontaneous regeneration of injured central axons. A 38-year-old man sustained traumatic transection of the thoracic spinal cord at upper vertebral level Th9. At 21 months after injury, the patient presented symptoms of a clinically complete spinal cord injury (American Spinal Injury Association class A-ASIA A). One of the patient's olfactory bulbs was removed and used to derive a culture containing olfactory ensheathing cells and olfactory nerve fibroblasts. Following resection of the glial scar, the cultured cells were transplanted into the spinal cord stumps above and below the injury and the 8-mm gap bridged by four strips of autologous sural nerve. The patient underwent an intense pre- and postoperative neurorehabilitation program. No adverse effects were seen at 19 months postoperatively, and unexpectedly, the removal of the olfactory bulb did not lead to persistent unilateral anosmia. The patient improved from ASIA A to ASIA C. There was improved trunk stability, partial recovery of the voluntary movements of the lower extremities, and an increase of the muscle mass in the left thigh, as well as partial recovery of superficial and deep sensation. There was also some indication of improved visceral sensation and improved vascular autoregulation in the left lower limb. The pattern of recovery suggests functional regeneration of both efferent and afferent long-distance fibers. Imaging confirmed that the grafts had bridged the left side of the spinal cord, where the majority of the nerve grafts were implanted, and neurophysiological examinations confirmed the restitution of the integrity of the corticospinal tracts and the voluntary character of recorded muscle contractions. To our knowledge, this is the first clinical indication of the beneficial effects of transplanted autologous bulbar cells

Bioprocessing strategies to enhance the challenging isolation of neuro-regenerative cells from olfactory mucosa

Olfactory ensheathing cells (OECs) are a promising potential cell therapy to aid regeneration. However, there are significant challenges in isolating and characterizing them. In the current study, we have explored methods to enhance the recovery of cells expressing OEC marker p75NTR from rat mucosa. With the addition of a 24-hour differential adhesion step, the expression of p75NTR was significantly increased to 73 ± 5% and 46 ± 18% on PDL and laminin matrices respectively. Additionally, the introduction of neurotrophic factor NT-3 and the decrease in serum concentration to 2% FBS resulted in enrichment of OECs, with p75NTR at nearly 100% (100 ± 0% and 98 ± 2% on PDL and laminin respectively), and candidate fibroblast marker Thy1.1 decreased to zero. Culturing OECs at physiologically relevant oxygen tension (2–8%) had a negative impact on p75NTR expression and overall cell survival. Regarding cell potency, co-culture of OECs with NG108-15 neurons resulted in more neuronal growth and potential migration at atmospheric oxygen. Moreover, OECs behaved similarly to a Schwann cell line positive control. In conclusion, this work identified key bioprocessing fundamentals that will underpin future development of OEC-based cell therapies for potential use in spinal cord injury repair. However, there is still much work to do to create optimized isolation methods

Evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through short sequence duplications

Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (� 2 μg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera

Mesenchymal Stem Cell Graft Improves Recovery after Spinal Cord Injury in Adult Rats through Neurotrophic and Pro-Angiogenic Actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue

Neuron-glia cross talk in rat striatum after transient forebrain ischemia

Striatum is highly vulnerable to transient forebrain ischemia induced by the 4 vessel occlusion (4V0) method (Brierley 1976. Pulsinelli et al. 1982, Zini et al. 1990a). Massive degeneration and loss of Nissl-stained neurons occur within 24 hr from an ischemia of long duration (30 min) (Pulsinelli et al. 1982). Neuronal loss is mainly restricted to the lateral part of caudate-putamen (Pulsinelli et al. 1982, Zini et al. 1990a). Cellular alterations include loss of medium-size spiny projection neurons (Pulsinelli et al. 1982, Francis and Pulsinelli 1982), largely corresponding to dopaminoceptive neurons (Benfenati et al. 1989, Zoli et al. 1989), and increase in reactive astrocytes (Pulsinelli et al. 1982, Grimaldi et al. 1990) and microglia (Gehrmann et al. 1982). On the other hand, large cholinergie (Francis and Pulsinelli 1982) and medium-size aspiny somatostatin (SS)/neuropeptide Y (NPY)-containing interneurons are resistant to the ischemic insult (Pulsinelli et al. 1982, Grimaldi et al. 1990). In a few instances, such as in the case of SS and NPY immunoreactivity (IR), the initial loss is followed by full recovery within 7 (SS) or 40 (NPY) days post-ischemia (Grimaldi et al. 1990). However, it is not known whether some kind of recovery is present for the bulk of medium-size spiny projections neurons after the first days post-ischemia

Olfactory Ensheathing Cell Transplantation in Experimental Spinal Cord Injury:Effect size and Reporting Bias of 62 Experimental Treatments: A Systematic Review and Meta-Analysis

Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies (62 experiments, 1,164 animals) in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8-29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3-5). In conclusion, OEC transplantation exerts considerable beneficial effects on neurobehavioral recovery after traumatic experimental SCI. Publication bias was minimal and affirms the translational potential of efficacy, but safety cannot be adequately assessed. The data justify OECs as a cellular substrate to develop and optimize minimally invasive and safe cellular transplantation paradigms for the lesioned spinal cord embedded into state-of-the-art Phase I/II clinical trial design studies for human SCI