Cultured OEC versus cultured SC and versus native OEC
ABSTRACT: This microarray study aimed at identifying the differences in the global gene expression growth program of adult cultured olfactory ensheathing cells (cOEC) (from the olfactory bulb) versus adult cultured Schwann cells (SC) (from the sciatic nerve) and versus adult OEC directly dissected from the olfactory nerve layer (nOEC). The aim of the comparison between cOEC and SC is to define intrinsic molecular differences that distinguish cOEC from SC (both cell types support neuronal regeneration). The aim of the comparison between cOEC and nOEC is to determine the transcriptional responses that are induced in OEC during culturing. Overall design: Three-condition experiment: cOEC vs. SC, cOEC vs. nOEC, SC vs. nOEC. Biological replicates: 3 cOEC-SC replicates, 3 cOEC-nOEC replicates, 3 SC-nOEC replicates. A factorial design was used consisting of direct comparisons between the three cell types (Glonek and Solomon, 2004 (PMID 14744830).
INSTRUMENT(S): Agilent-011868 Rat Oligo Microarray G4130A (Feature Number version)
Project description:This microarray study aimed at identifying the differences in the global gene expression growth program of adult cultured olfactory ensheathing cells (cOEC) (from the olfactory bulb) versus adult cultured Schwann cells (SC) (from the sciatic nerve) and versus adult OEC directly dissected from the olfactory nerve layer (nOEC). The aim of the comparison between cOEC and SC is to define intrinsic molecular differences that distinguish cOEC from SC (both cell types support neuronal regeneration). The aim of the comparison between cOEC and nOEC is to determine the transcriptional responses that are induced in OEC during culturing. Three-condition experiment: cOEC vs. SC, cOEC vs. nOEC, SC vs. nOEC. Biological replicates: 3 cOEC-SC replicates, 3 cOEC-nOEC replicates, 3 SC-nOEC replicates. A factorial design was used consisting of direct comparisons between the three cell types (Glonek and Solomon, 2004 (PMID 14744830).
Project description:The remarkable feature of Schwann cells (SCs) to transform into a repair phenotype turned the spotlight on this powerful cell type. SCs provide the regenerative environment for axonal re-growth after peripheral nerve injury (PNI) and play a vital role in differentiation of neuroblastic tumors into a benign subtype of neuroblastoma, a tumor originating from neural crest-derived neuroblasts. Hence, understanding their mode-of-action is of utmost interest for new approaches in regenerative medicine, but also for neuroblastoma therapy. However, literature on human SCs is scarce and it is unknown to which extent human SC cultures reflect the SC repair phenotype developing after PNI in patients. We performed high-resolution proteome profiling and RNA-sequencing on highly enriched human SC and fibroblast cultures, control and ex vivo degenerated nerve explants to identify novel molecules and functional processes active in repair SCs. In fact, we found cultured SCs and degenerated nerves to share a similar repair SC-associated expression signature, including the upregulation of JUN, as well as two prominent functions, i.e., myelin debris clearance and antigen presentation via MHCII. In addition to myelin degradation, cultured SCs were capable of actively taking up cell-extrinsic components in functional phagocytosis and co-cultivation assays. Moreover, in cultured SCs and degenerated nerve tissue MHCII was upregulated at the cellular level along with high expression of chemoattractants and co-inhibitory rather than -stimulatory molecules. These results demonstrate human SC cultures to execute an inherent program of nerve repair and support two novel repair SC functions, debris clearance via phagocytosis-related mechanisms and type II immune-regulation. Overall design: mRNA of 27 samples were sequenced (50bp, single end) and analyzed. Biological replicates were performed.
Project description:Spiral ganglion neurons (SGNs) and the associated components of the auditory nerve are primary carriers of auditory information from hair cells to the brain. Loss of SGNs occurs with many pathological conditions, resulting in permanent sensorineural hearing loss. Neural stem/progenitors (NSPs) have been well-characterized in several locations of adult brain and retina. However, it is unclear whether NSPs are present in the adult auditory nerve. Here we examined the self-renewal potential of the adult auditory nerve using ouabain application as a well-established mouse model of acute SGN injury. The observed increase in cell proliferation, alteration in enchromatin/heterochromatin ratio and down-regulation of histone deacetylase expression in glial cells suggest that the quiescent glial cells convert to an activated state after SGN degeneration. This was further confirmed by global gene expression analysis of injured auditory nerves, which showed up-regulation of numerous neurogenesis- and/or development-associated genes shortly after ouabain exposure. These genes include molecular markers commonly used for the identification of NSPs. Under a strict culture regimen, auditory nerve-derived cells of adult mouse ears gave rise to neurospheres, suggesting that multipotent NSPs are present in adult cochlear nerve. Neurosphere assays on Sox2 transgenic mice revealed that Sox2+ glial cells are the source for NSPs. Our data also showed that acute injury or hypoxia enhances neurosphere formation. Taken together, our study revealed that glial cells of adult cochlea exhibit several NSP characteristics, and hence these mature non-neuronal cells may be important targets for promoting self-repair of degenerative auditory nerves. Auditory nerves were removed from the temporal bones of adult CBA/CaJ mice, aged 8 to 12 weeks. Tissues were either collected and used directly as the tissue samples or dissociated and used for the cell culture samples. Dissociated auditory nerve cells were propagated and grown to full confluency (5-7 days), constituting the cultured cell samples. For neurosphere samples, growth medium was changed to neurosphere formation medium and the cells were cultured for an additional 12 days. All samples were prepared in triplicate (n=3).
Project description:The aim of this study was to use global gene expression profiling to define intrinsic molecular differences that distinguish olfactory ensheathing cells from mucosa (OM-OECs) from olfactory ensheathing cells from olfactory bulb (OB-OECs). 10,000 OECs from olfactory mucosa (OM) or olfactory bulb (OB) were isolated from 4 rats. Overall design: Two-color experiment, OM (Cy5) vs. OB (Cy3) OECs. Biological replicates: 4. Paired samples.
Project description:Gene expression analysis of 2-month-old Ctrl and Tfam-SCKO mice. At this age mitochondrial function is disrupted in the Schwann cells of Tfam-SCKO mice ,but their nerves display only very limited pathology. Mitochondrial dysfunction is a common cause of peripheral neuropathy. Much effort has been devoted to examining the role played by neuronal/axonal mitochondria, but how mitochondrial deficits in peripheral nerve glia (Schwann cells, SCs) contribute to peripheral nerve diseases remains unclear. Here, we investigate a mouse model of peripheral neuropathy secondary to SC mitochondrial dysfunction (Tfam-SCKOs). We show that disruption of SC mitochondria activates a maladaptive integrated stress response through actions of heme-regulated inhibitor kinase (HRI), and causes a shift in lipid metabolism away from fatty acid synthesis toward oxidation. These alterations in SC lipid metabolism result in depletion of important myelin lipid components as well as in accumulation of acylcarnitines, an intermediate of fatty acid b-oxidation. Importantly, we show that acylcarnitines are released from SCs and induce axonal degeneration. A maladaptive integrated stress response as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies. Total RNA samples were prepared by isolating and pooling RNA from three different 2-month-old MPZ-Tfam KO and Ctrl mice. 2 replicates per genotype were used in this experiment and they were prepared entirely independently.
Project description:The olfactory system is a part of the mammalian central nervous system and is characterized by neurogenesis and replacement of degenerated neurons. Clinically, it has been reported that the elderly has a high incidence of olfactory impairment. However, the molecular mechanisms underlying aging-associated defect of olfaction have yet been unclear. To elucidate this problem, we performed micoarray analysis against murine olfactory epithelium and compared the transcriptome of them between young-adult and aged mice. Overall design: Gene expression of olfactory epihtelium from young-adult (8 weeks of age) and aged (16 months of age) male C57BL/6 mice was measured. Each total RNA sample pooled from 5 biological replicates (two experimental replicate) was analyzed using microarray.
Project description:Comparison of three populations of OECs derived from the ONL of rats. The three populations were cultured employing the same culture medium but were maintained in vitro for a different amount of time (number of population doublings or PDs). OEC Ep cells were maintained for less than one week in vitro (less than 3 PDs); OEC Lp were maintained for over a year (more than 50 PDs) and TEG3 OEC cell line has been serially passed in culture for long-periods of time after genetic immortalization. Experiment Overall Design: RNA sample preparation and experimental design. Total RNA was isolated with TRIZOL (Invitrogen, Gibco-BRL) from primary OEC cultures (OEC Ep and Lp) and the OEC cell line (TEG3). Cultured cells were harvested at confluency with a rubber scrapper, resuspended in the TRIZOL reagent and processed following the manufacturers’ instructions. Two RNA pools were obtained from each OEC population (OEC Ep, OEC Lp and TEG3 cells). Each RNA pool was purified from three individual cultures (three 100mm dishes) and in the case of OEC Ep, each culture was derived from one rat. The total RNA was resuspended in DEPC (Sigma) water and stored at -70ºC prior to use. Total RNA concentration and purity were determined using Agilent2100 Bioanalizer. Experiment Overall Design: Microarray analysis. Total RNA pools were cleaned using Rneasy Kit from Qiagen. Biotinylated cRNA probes were generated from each RNA pool, fragmented, and applied to two different sets of Rat Genome Affymetrix GeneChip U34A Array. Affymetrix software (Microarray Suite version 5.0) was used to filter inaccurately represented probe sets.