Olfactory ensheathing cells (OECs) are a unique type of glia with common properties of astrocyte and Schwann cells. Cultured OECs have two morphological phenotypes, astrocyte-like OECs and Schwann cell-like OECs. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of these reversible changes is still unknown. The aim of this paper is to establish a method for the morphology plasticity of cultured OECs, and investigate the underlying mechanism. Using the primary culture of OECs and immunocytochemistry, the morphology of OECs was observed under serum, serum free media or dB-cAMP drug treatment. Statistical analysis was performed to test differences among the percentages of OEC subtypes under these conditions. The results showed that under serum free media, (95.2±3.7)% of OECs showed Schwann cell-like morphology, and (4.8±3.7)% of OECs showed astrocyte-like morphology; however, under 10% serum media, (42.5±10.4)% of OECs exhibited Schwann cell-like morphology, and (57.5±10.4)% of OECs exhibited astrocyte-like morphology. When media was changed back to serum free media for 24 h, (94.8±5.0)% of OECs showed Schwann cell-like morphology, and (5.2±5.0)% of OECs showed astrocyte-like morphology. Furthermore, culture condition with or without serum did not affect the expression of OEC cell marker, p-75 and S-100. Finally, dB-cAMP, an analog of cAMP, through inhibiting the formation of F-actin stress fibers and focal adhesion, induced the morphology switch from astrocyte-like to Schwann cell-like morphology under serum condition, promoted the branches and the growth of processes. These results suggest that serum induces the morphology plasticity of cultured OECs, which is mediated by cytoplasmic cAMP level through regulating the formation of F-actin stress fibers and focal adhesion.
Animals ; Astrocytes ; cytology ; physiology ; Cells, Cultured ; Culture Media ; pharmacology ; Cyclic AMP ; physiology ; Male ; Neuroglia ; cytology ; physiology ; Olfactory Bulb ; cytology ; physiology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; physiology ; Serum ; physiology

OBJECTIVETo investigate the phenotypic, molecular and biological characteristics of adipose tissue-derived stromal cells (ADSCs) differentiated alonely a Schwann cells (SCs) lineage and to provide a new cells' seed source for nerve tissue engineering or cell therapy.

METHODSCultured ADSCs were isolated from SD rats and the undifferentiated ADSCs were confirmed by detection of MSC-specific cell-surface markers. The ADSCs were differentiated along a glial cell lineage using an established cocktail of growth factors. Following differention, we used immunofluorescene staining and RT-PCR to evaluate the characteristics of differentiated WJMSCs.

RESULTSADSCs were successfully isolated from the rats' fat tissue. The isolated ADSCs expressed CD29, CD90 but not CD34, CD44 nor CD45. Osteogenic differentiation was detected by Alizarin red staining and adipogenic differentiation was comfirmed by Oil-red O staining. ADSCs treated with a mixture of glial growth factors adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and RT-PCR analysis revealed that the treated cells expressed the glial markers S100, P75 and glial fibrillary acidic protein indicative of differentiation.

CONCLUSIONADSCs can be differentiated into cells that are Schwann-like in terms of morphologic features and phenotype and could be suitable Schwann-cell substitutes for nerve repair in clinical applications.

Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology

AIMTo compare the effects of primary culture Schwann cells and cryopreserved Schwann cells on the injured peripheral nerve regeneration.

METHODSThe primary culture Schwann cells and cryopreserved Schwann cells were transplanted into the silicone tubes that bridged the resected sciatic nerves. At different time after transplantation, HRP was injected into the sciatic nerve trunks at the distal end of the silicone tubes, and then the HRP labeled neurons in dorsal root ganglions and anterior horns of spinal cord were counted. The complex action potential of regenerated nerve fibers was measured. Myelination on regenerated nerve fibers was investigated under electronic microscope.

RESULTSAt different time after transplantation, there were not significant differences that the HRP labeled neurons in dorsal root ganglions and anterior horns of spinal cord, the complex action potential of regenerated nerve fibers and myelination on regenerated nerve fibers (P > 0.05), between primary culture and cryopreserved Schwann cells.

CONCLUSIONThe cryopreserved Schwann cells still play the same important role in the regeneration of injured peripheral nerves as the primary culture Schwann cells do.

Action Potentials ; Animals ; Cells, Cultured ; Cryopreservation ; Nerve Regeneration ; physiology ; Peripheral Nerves ; physiology ; Primary Cell Culture ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; physiology

OBJECTIVETo test a nerve bridge substitute for peripheral nerve repair by tissue-engineering approach.

METHODSAn artificial nerve fabricated with a scroll of amnion derivative (ZQ membrane) and cultured autogenous Schwann cell was sutured to bridge sciatic nerve defect of 2.5 cm in length in rats. The specimens were assessed with tracking study, histology, electrophysiological technique, NF200, and synaptophysin-38 (SYP) immuno histochemical staining 3 months postoperatively.

RESULTSThe regenerated nerve sprouted 3 months after the operation. The regenerated nerve fibers were plentiful and could grow into the recipient nerve and target muscle's motor end plate (MEP) areas to reinnervate target muscle, and reconstruct function of nerve-muscle junction. Functional recovery could reach to 40%-60% of normal control. Nerve-muscle conduction velocity (N-MCV) arrived at 21.77 +/- 1.15 m/s.

CONCLUSIONSA tissue engineering material fabricated with a scroll of ZQ membrane and cultured autologous Schwann cell may be a useful substitute for nerve repair.

Amnion ; cytology ; Animals ; Cells, Cultured ; Female ; Male ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; Sciatic Nerve ; injuries ; surgery ; Tissue Engineering ; methods

OBJECTIVETo explore the factors which induce differentiation of embryonic neural stem cells.

METHODSRat embryonic neural stem cells were co-cultured with newborn rat Schwann cells in serum-free medium. The phenotype and specific-markers including tubulin-beta, glial fibrillary acidic protein (GFAP) and galactorcerebroside (GalC), were demonstrated by phase contrast microscopy and double immunofluorescence staining.

RESULTSOverall, 80% +/- 5% of neural stem cells protruded several elongated processes and expressed tubulin-beta antigen at high levels, while 20 +/- 3% of them protruded several short processes and were GalC or GFAP positive.

CONCLUSIONThe factors secreted by Schwann cells could induce rat embryonic neural stem cell to differentiate.

Animals ; Cell Differentiation ; Coculture Techniques ; Embryo, Mammalian ; cytology ; Fluorescent Antibody Technique ; Microscopy, Phase-Contrast ; Nerve Growth Factors ; physiology ; Neurons ; cytology ; Phenotype ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; physiology ; Stem Cells ; cytology

BACKGROUNDVarious tissue engineering strategies have been developed to facilitate axonal regeneration after spinal cord injury. This study aimed to investigate whether neural stem cells (NSCs) could survive in poly(L-lactic-co-glycolic acid) (PLGA) scaffolds and, when cografted with Schwann cells (SCs), could be induced to differentiate towards neurons which form synaptic connection and eventually facilitate axonal regeneration and myelination and motor function.

METHODSNSCs and SCs which were seeded within the directional PLGA scaffolds were implanted in hemisected adult rat spinal cord. Control rats were similarly injured and implanted of scaffolds with or without NSCs. Survival, migration, differentiation, synaptic formation of NSCs, axonal regeneration and myelination and motor function were analyzed. Student's t test was used to determine differences in surviving percentage of NSCs. One-way analysis of variance (ANOVA) was used to determine the differences in the number of axons myelinated in the scaffolds, the mean latency and amplitude of cortical motor evoked potentials (CMEPs) and Basso, Beattie & Bresnahan locomotor rating scale (BBB) score. The χ(2) test was used to determine the differences in recovery percentage of CMEPs.

RESULTSNSCs survived, but the majority migrated into adjacent host cord and died mostly. Survival rate of NSCs with SCs was higher than that of NSCs without SCs ((1.7831 ± 0.0402)% vs. (1.4911 ± 0.0313)%, P < 0.001). Cografted with SCs, NSCs were induced to differentiate towards neurons and might form synaptic connection. The mean number of myelinated axons in PLGA + NSCs + SCs group was more than that in PLGA + NSCs group and in PLGA group ((110.25 ± 30.46) vs. (18.25 ± 3.30) and (11.25 ± 5.54), P < 0.01). The percentage of CMEPs recovery in PLGA + NSCs + SCs group was higher than in the other groups (84.8% vs. 50.0% and 37.5%, P < 0.05). The amplitude of CMEPs in PLGA + NSCs + SCs group was higher than in the other groups ((1452.63 ± 331.70) µV vs. (428.84 ± 193.01) µV and (117.33 ± 14.40) µV, P < 0.05). Ipsilateral retransection resulted in disappearance again and functional loss of CMEPs for a few days. But contralateral retransection completely damaged the bilateral motor function.

CONCLUSIONSNSCs can survive in PLGA scaffolds, and SCs promote NSCs to survive and differentiate towards neurons in vivo which even might form synaptic connection. The scaffolds seeded with cells facilitate axonal regeneration and myelination and motor function recovery. But regenerating axons have limited contribution to motor function recovery.

Animals ; Axons ; physiology ; Cells, Cultured ; Electrophysiology ; Female ; Fluorescent Antibody Technique ; Lactic Acid ; chemistry ; Nerve Regeneration ; physiology ; Neural Stem Cells ; cytology ; Polyglycolic Acid ; chemistry ; Pregnancy ; Rats ; Rats, Wistar ; Schwann Cells ; cytology ; Spinal Cord Injuries ; therapy ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVETo investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells.

METHODSBone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry.

RESULTSMononuclear cells separated by Percoll's were passaged 10 times by trypsin/ethylenediaminetetraacetic acid (EDTA) digestion in 40 days, and BMSCs increased about 6x10(7) times in this short period. Immunohistochemistry identified that BMSCs were CD34- and CD31-, but they expressed neuron specific enolase; 0.01%-0.02% of total cells expressed nestin, the marker for neural progenitor cells; 40%-50% cells stained heavily by neurofilament 200; and no glial fibrillary acidic protein (GFAP) positive cells were identified; S100 expression was detected among 0.1%-0.2% cells.

CONCLUSIONSBone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; physiology ; Cell Proliferation ; Humans ; Immunohistochemistry ; Intermediate Filament Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurofilament Proteins ; metabolism ; Phosphopyruvate Hydratase ; metabolism ; S100 Proteins ; metabolism ; Schwann Cells ; cytology

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Animals ; Axotomy ; Cell Movement ; Cell Proliferation ; Male ; Membrane Glycoproteins/*physiology ; Membrane Proteins/pharmacology ; *Nerve Regeneration ; Nerve Tissue Proteins/pharmacology ; Neurites/drug effects/*physiology/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/pharmacology ; Schwann Cells/cytology/*physiology ; Sensory Receptor Cells/*physiology

OBJECTIVETo investigate the effects of Ginsenoside Rb(1) on the proliferation of Schwann cells in culture.

METHODSApplying MTT assay and Thymidine incorporation assay, the effects of Ginsenoside Rb(1) on the proliferation of Schwann cells isolated from the sciatic nerve of adult rat were studied.

RESULTSGinsenoside Rb(1) (10 microg/ml) significantly induced Schwann cell proliferation, the effect was similar to NGF (50 microg/ml). At high concentrations of Ginsenoside Rb(1) (1 mg/ml), the proliferation of Schwann cells was significantly inhibited.

CONCLUSIONSGinsenoside Rb(1) at the optimal concentrations is found to be effective in inducing the proliferation of Schwann cells, but at higher concentrations the drug is cytotoxic for Schwann cells.

Animals ; Cell Division ; drug effects ; physiology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Ginsenosides ; adverse effects ; pharmacology ; Immunohistochemistry ; Nerve Regeneration ; drug effects ; Probability ; Rats ; Rats, Inbred Strains ; Schwann Cells ; drug effects ; physiology ; Sciatic Nerve ; cytology ; physiology ; Sensitivity and Specificity

OBJECTIVETo investigate the effect of mitogen-activated protein kinase (MEK) kinase cascade, extracellular signal-regulated kinase (ERK1/2) signal pathway on Schwann cells proliferation promoted by Pyrroloquinoline Quinine (PQQ) and its molecular mechanisms.

METHODSSchwann cells were cultured and purified in vitro. The purity was identified by S-100. Different time and concentration of PQQ was added into culture medium. The expression of ERK1/2 and phosphorylated-ERK1/2 was detected by western blot. The expression of p-ERK1/2 after blocking of MEK signal pathway by specific inhibitor PD98059 was detected by western blot.

RESULTSMorphological change was observed in PQQ treated Schwann cells. 1-500 nmol/L PQQ could up-regulate the expression of p-ERK1/2, and 1000 nmol/L had no effects, while 10 000 nmol/L exhibited inhibitory effect (P < 0.05). p-ERK1/2 increased to peak 1 h after PQQ added, and this up-regulation of p-ERK1/2 was inhibited by PD98059 (P < 0.05).

CONCLUSIONSPQQ could affect morphology of Schwann cells and activation of ERK1/2. MEK inhibitor PD98059 could, block this activation. It suggests that MEK/ERK signal pathway should be involved in Schwann cells proliferation promoted by PQQ.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; physiology ; Mitogen-Activated Protein Kinases ; metabolism ; physiology ; Pyrroles ; pharmacology ; Quinolines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; drug effects ; Signal Transduction