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The massive loss of oligodendrocytes caused by various pathological factors is a basic feature of many demyelinating diseases of the central nervous system (CNS). Based on a variety of studies, it is now well established that impairment of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is a vital event in the failed treatment of demyelinating diseases. Recent evidence suggests that Foxg1 is essential for the proliferation of certain precursors and inhibits premature neurogenesis during brain development. To date, very little attention has been paid to the role of Foxg1 in the proliferation and differentiation of OPCs in demyelinating diseases of the CNS. Here, for the first time, we examined the effects of Foxg1 on demyelination and remyelination in the brain using a cuprizone (CPZ)-induced mouse model. In this work, 7-week-old Foxg1 conditional knockout and wild-type (WT) mice were fed a diet containing 0.2% CPZ w/w for 5 weeks, after which CPZ was withdrawn to enable remyelination. Our results demonstrated that, compared with WT mice, Foxg1-knockout mice exhibited not only alleviated demyelination but also accelerated remyelination of the demyelinated corpus callosum. Furthermore, we found that Foxg1 knockout decreased the proliferation of OPCs and accelerated their differentiation into mature oligodendrocytes both in vivo and in vitro. Wnt signaling plays a critical role in development and in a variety of diseases. GSK-3β, a key regulatory kinase in the Wnt pathway, regulates the ability of β-catenin to enter nuclei, where it activates the expression of Wnt target genes. We then used SB216763, a selective inhibitor of GSK-3β activity, to further demonstrate the regulatory mechanism by which Foxg1 affects OPCs in vitro. The results showed that SB216763 clearly inhibited the expression of GSK-3β, which abolished the effect of the proliferation and differentiation of OPCs caused by the knockdown of Foxg1. These results suggest that Foxg1 is involved in the proliferation and differentiation of OPCs through the Wnt signaling pathway. The present experimental results are some of the first to suggest that Foxg1 is a new therapeutic target for the treatment of demyelinating diseases of the CNS.
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BACKGROUND: Oligodendrocyte precursor cell transplantation is one of the keys to the treatment of white matter damage in premature infants. At present, there is a lack of research on the comparison of oligodendrocyte precursor cells induced by neural stem cells derived from human fetal brain cultured in vitro in different vessels worldwide. OBJECTIVE: To observe the morphology of human oligodendrocyte progenitor cells and pre-oligodendrocytes in different cell culture vessels (6-well plates, 24-well plates and T25 flasks). METHODS: The 6-well plates, 24-well plates and T25 flasks were used as culture vessels to culture human oligodendrocyte progenitor cells and pre-oligodendrocytes. The characteristics of human oligodendrocyte progenitor cells were identified by immunofluorescence staining and flow cytometry. The morphology of cells was observed by an ordinary light microscope. Cell counts were performed according to cell morphology and statistical analysis was performed. RESULTS AND CONCLUSION: (1) The oligodendrocyte progenitor cell body was round and the bipolar protrusions were bead-like; the pre-oligodendrocyte protrusions were more than two poles, and did not bifurcate. (2) The ratios of oligodendrocyte progenitor cell morphology in the oligodontia lines were significantly higher in the 6-well plates than those in the 24-well plates and T25 flasks (P < 0.05), followed by T25 flasks and 24-well plates. Morphological ratios of pre-oligodendrocytes were significantly higher in the 24-well plates compared to the 6-well plates and T25 flasks (P < 0.01), followed by T25 flasks and 6-well plates. (3) The cells cultured in the 6-well plate had fewer dregs, and the morphology, vigor and growth were better than those of the other culture vessels. (4) According to morphological view, 6-well plates are more suitable for oligodendrocyte progenitor cell growth, and 24-well plates are more suitable for pre-oligodendrocytes growth.
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Objective@#To observe the effect of quercetin on myelin regeneration in desyelinating mice induced by cuprizone (CPZ).@*Methods@#A total of 50 C57BL/6J mice were randomly divided into normal control group, model group, low, medium and high dose quercetin groups, 10 mice in each group. In addition to the normal control group, mice demyelinating model was induced by feeding with 0.2% CPZ rodent feed. Quercetin solution was administered to the low, medium and high dose quercetin groups at 25, 50 and 100 mg/kg, and to the normal control group and the model group at 1 time/d. After 5 weeks of continuous gavage, the weight of mice was recorded every week, and the experiment of rotating bar was carried out. After 5 weeks, the changes of the myelin sheath in the corpus callosum of mice were observed by luxol fast blue (LFB) and transmission electron microscope (TEM). Immunofluorescence method was used to determine the protein expressions of myelin basic protein (MBP) and oligodendrocyte transcription 2actor (Olig2) in mouse brain tissue. The expression of MBP and cyclic nucleotide-3'phosphate hydrolase (CNPase) in mouse corpus callosum was determined by Western blot.@*Results@#After 2-5 weeks, compared with the model group, the body mass of the medium and high dose quercetin groups significantly increased (P<0.01), and the score of turning rod significantly increased (P<0.01). The LFB observation showed that the demyelination score of corpus callosum in low, medium and high dose quercetin groups (2.23 ± 0.25, 1.50 ± 0.15, 1.14 ± 0.97 vs. 2.83 ± 0.18) significantly decreased (P<0.01). TEM observation showed that the G-ratio value in low, medium and high dose quercetin groups (0.75 ± 0.05, 0.75 ± 0.08, 0.73 ± 0.08 vs. 0.87 ± 0.05) significantly reduced (P<0.01). Immunofluorescence observation showed that the positive expression of MBP (37.40 ± 2.41, 37.40 ± 1.14 vs. 24.40 ± 3.65) and Olig2 (7.40 ± 1.14, 4.60 ± 1.14 vs. 2.80 ± 0.84) in the medium and high dose quercetin groups significantly increased (P<0.05). WB showed that the expression of MBP protein (1.32 ± 0.12, 0.80 ± 0.34 vs. 0.21 ± 0.07) and CNPase protein (0.72 ± 0.13, 1.06 ± 0.36 vs. 0.36 ± 0.21) in the medium and high dose quercetin groups significantly increased (P<0.05).@*Conclusions@#Quercetin (50-100 mg/kg) can reduce myelin injury and promote myelin regeneration in CPZ mice, as a neuroprotective effect on CPZ mice.
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OBJECTIVE@#To observe the effect of electroacupuncture (EA) at "Jiaji" (EX-B 2) points on the proliferation and differentiation of oligodendrocyte precursor cells in rats with acute incomplete spinal cord injury, and to explore the mechanism of EA on improving motor function of spinal cord injury.@*METHODS@#A total of 72 male SPF SD rats were randomly divided into a sham operation group, a model group, an EA group and a medication group, 18 rats in each group. Each group was further divided into 1-day subgroup, 7-day subgroup and 14-day subgroup, 6 rats in each subgroup. The T acute incomplete spinal cord injury model was established by modified Allen's method in the model group, EA group and medication group. The rats in each group received intraperitoneal injection of 5-bromodeoxyuridine (BrdU, 50 mg/kg), once a day, and each subgroup received continuous injection for 1, 7, 14 times for cell proliferation labeling. The rats in the EA group were treated with EA at "Jiaji" (EX-B 2) points 3-4 mm next the spinous process of the upper and lower segments of the injured spinal cord (T, T) with a frequency of 2 Hz/100 Hz and intensity of 1-2 mA. The muscle twitch at the treatment site was taken as the degree. The treatment was given 20 min each time, once a day. In the medication group, monosialogangliosides (GM1) was injected intraperitoneally (10 mg/kg), once a day. The subgroups of EA group and medication group were treated for 1, 7, 14 times. The score of Basso Beattie Bresnahan (BBB) was used to evaluate the motor function of hind limbs. The co-expression of BrdU/NG2 positive cells was detected by immunofluorescence, and the expression of Olig2 and Sox10 was detected by Western blot.@*RESULTS@#Compared with the sham operation group, the BBB score was decreased 1 day, 7 days and 14 days after operation in the model group (<0.05), the expression of Olig2 and Sox10 was increased (<0.05), and the co-expression of BrdU/NG2 positive cells was increased 7 days and 14 days after operation (<0.05). Seven days and 14 days after operation, the BBB score in the EA group and medication group was higher than that in the model group (<0.05), and the co-expression of BrdU/NG2 in the medication group was higher than that in the model group (<0.05). Fourteen days after operation, the co-expression of BrdU/NG2 in the EA group was higher than that in the model group (<0.05); 1 day, 7 days and 14 days after operation, the expression of Olig2 and Sox10 in the EA group and medication group was higher than that in the model group (<0.05). Compared with the medication group, the co-expression of BrdU/NG2 positive cells in the EA group 14 days after operation was decreased (<0.05); 1 day, 7 days and 14 days after operation, the expression of Olig2 and Sox10 in the EA group was decreased (<0.05).@*CONCLUSION@#EA at "Jiaji" (EX-B 2) points could promote the expression of Olig2 and Sox10 after spinal cord injury, which has similar effects with GM1. It could promote the proliferation and differentiation of oligodendrocyte precursor cells into oligodendrocytes, so as to promote the recovery of motor function of rats.
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Animals , Humans , Male , Rats , Acupuncture Points , Cell Differentiation , Cell Proliferation , Electroacupuncture , Oligodendrocyte Precursor Cells , Cell Biology , Oligodendrocyte Transcription Factor 2 , Metabolism , Random Allocation , Rats, Sprague-Dawley , SOXE Transcription Factors , Metabolism , Spinal Cord , Spinal Cord Injuries , TherapeuticsABSTRACT
Glioblastoma (GBM) is the most common and lethal primary neoplasm in the central nervous system. Despite intensive treatment, the prognosis for patients with GBM remains poor, with a median survival of 14-16 months. 90% of GBMs are primary GBMs that are full-blown at diagnosis without evidences of a pre-existing less-malignant precursor lesion. Therefore, identification of the cell(s) of origin for GBM-the normal cell or cell type that acquires the initial GBM-promoting genetic hit(s)-is the key to the understanding of the disease etiology and the development of novel therapies. Neural stem cells and oligodendrocyte precursor cells are the two major candidates for the cell(s) of origin for GBM. Latest data from human samples have reignited the longstanding debate over which cells are the clinically more relevant origin for GBMs. By critically analyzing evidences for or against the candidacy of each cell type, we highlight the most recent progress and debate in the field, explore the clinical implications, and propose future directions toward early diagnosis and preventive treatment of GBMs.
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The obstacle to successful remyelination in demyelinating diseases, such as multiple sclerosis, mainly lies in the inability of oligodendrocyte precursor cells (OPCs) to differentiate, since OPCs and oligodendrocyte-lineage cells that are unable to fully differentiate are found in the areas of demyelination. Thus, promoting the differentiation of OPCs is vital for the treatment of demyelinating diseases. Shikimic acid (SA) is mainly derived from star anise, and is reported to have anti-influenza, anti-oxidation, and anti-tumor effects. In the present study, we found that SA significantly promoted the differentiation of cultured rat OPCs without affecting their proliferation and apoptosis. In mice, SA exerted therapeutic effects on experimental autoimmune encephalomyelitis (EAE), such as alleviating clinical EAE scores, inhibiting inflammation, and reducing demyelination in the CNS. SA also promoted the differentiation of OPCs as well as their remyelination after lysolecithin-induced demyelination. Furthermore, we showed that the promotion effect of SA on OPC differentiation was associated with the up-regulation of phosphorylated mTOR. Taken together, our results demonstrated that SA could act as a potential drug candidate for the treatment of demyelinating diseases.
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Animals , Female , Rats , Apoptosis , Cell Differentiation , Cell Proliferation , Cells, Cultured , Demyelinating Diseases , Encephalitis , Encephalomyelitis, Autoimmune, Experimental , Mice, Inbred C57BL , Myelin Basic Protein , Metabolism , Neuroprotective Agents , Oligodendrocyte Precursor Cells , Metabolism , Remyelination , Shikimic Acid , TOR Serine-Threonine Kinases , MetabolismABSTRACT
Objective To investigate the effect of VEGF on proliferation and migration of oligodendrocyte precursor cells. Methods To isolate and culture of oligodendrocyte precursor cells from mice. VEGF acts on the oligodendrocyte precursor cells for 48 h, meanwhile, the control group was set up and treated without VEGF. The proliferation of cells was detected by MTT assay, the cell migration was detected with a Boyden chamber, the levels of MMP-9, MMP-2, β-catenin, C-myc, cyclin D1 proteins in the cells were detected by western blotting. Wnt/β-catenin signaling pathway activator LiCl treated the oligodendrocyte precursor cells for 48 h, and the cell proliferation and migration were detected. Results The survival rate and number of migrated oligodendrocyte precursor cells were significantly higher than those of the control group after treated with VEGF (P＜ 0. 01). The levels of MMP-9, MMP-2, β-catenin, C-myc and cyclin D1 in the oligodendrocyte precursor cells after treated with VEGF were significantly higher than those in the control group (P＜ 0. 01). The cell proliferation and migration after treated with Wnt/β-catenin signaling activator LiCl were consistent with the proliferation and migration of cells after treated with VEGF. Conclusions VEGF promotes proliferation and migration of oligodendrocyte precursor cells. The mechanism of action is related to Wnt/β-catenin signaling pathway.
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The differentiation and maturation of oligodendrocyte precursor cells (OPCs) is essential for myelination and remyelination in the CNS. The failure of OPCs to achieve terminal differentiation in demyelinating lesions often results in unsuccessful remyelination in a variety of human demyelinating diseases. However, the molecular mechanisms controlling OPC differentiation under pathological conditions remain largely unknown. Myt1L (myelin transcription factor 1-like), mainly expressed in neurons, has been associated with intellectual disability, schizophrenia, and depression. In the present study, we found that Myt1L was expressed in oligodendrocyte lineage cells during myelination and remyelination. The expression level of Myt1L in neuron/glia antigen 2-positive (NG2) OPCs was significantly higher than that in mature CC1 oligodendrocytes. In primary cultured OPCs, overexpression of Myt1L promoted, while knockdown inhibited OPC differentiation. Moreover, Myt1L was potently involved in promoting remyelination after lysolecithin-induced demyelination in vivo. ChIP assays showed that Myt1L bound to the promoter of Olig1 and transcriptionally regulated Olig1 expression. Taken together, our findings demonstrate that Myt1L is an essential regulator of OPC differentiation, thereby supporting Myt1L as a potential therapeutic target for demyelinating diseases.
Subject(s)
Animals , Mice , Cell Differentiation , Physiology , Demyelinating Diseases , Lysophosphatidylcholines , Toxicity , Mice, Inbred C57BL , Nerve Tissue Proteins , Metabolism , Oligodendrocyte Precursor Cells , Cell Biology , Metabolism , Oligodendroglia , Cell Biology , Metabolism , Remyelination , Physiology , Transcription Factors , MetabolismABSTRACT
Demyelinating diseases are a group of nervous system disorders characterized by myelin sheath damage. Demyelinating diseases can seriously affect the quality of life of its victims and still lack satisfying therapeutic options. Oligodendrocyte precursor cells (OPCs) are progenitor cells exist in the central nervous system (CNS), with migration and proliferation capacities and potential to differentiate into oligodendrocytes (OLs), which are myelinated cells in the CNS, indicating that OPCs are closely related to myelination and post-injury regeneration in CNS. Recently, with the improved understanding of the mechanisms of OPCs development and lineage specification, the approaches to gain functional OPCs through directed differentiation from pluripotent stem cells or lineage reprogramming from somatic cells have been greatly promoted. Based on these achievements, OPCs transplantation becomes a promising therapeutic option for the treatment of demyelinating diseases of CNS. In this review, we summarized the latest research progress in this field.
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Objective Cell therapy is a possible effective way to treat myelination disorder diseases.Cell therapy needs to apply a method for culturing oligodendrocyte precursor cells.Therefore,this study was to develop a stable,efficient and economical method for obtaining human oligodendrocyte precursor cells (OPCs) in order to provide cell required for clinical treatment.And this will provide a new option for clinical applications.Methods Human OPCs were obtained through magnetic bead sorting and cultured in OPCs proliferation medium.For a short-time (2,4,6,8days) and long-time culture,morphology of OPCs was observed.The fourth generation of OPCs was analyzed for expression of OPCs specific markers O4,Soxl0 and platelet derived growth factor alpla receptors(PDGFαR) and the capacity to differentiate into oligodendrocytes by immunofluorescence staining.At the same time,the effects of B27 and N1 on OPCs growth state were inspected as well.Results For a short-time culture,OPCs had typical bipolar or tripolar morphology and proliferated in good condition.For a long-time culture,all 4 generations OPCs had typical bipolar or tripolar morphology;the fourth generation OPCs highly(＞ 90％) expressed 04,Sox10 and PDGFαR,after induction,OPCs could be differentiated into oligodendrocytes.After 4 generations of long-time culture,OPCs already maintained the original sharp,high purity and had the capacity to differentiate into oligodendrocytes.It was indicated that this culture system was suitable for human OPCs for a long-time culture.Conclusions Overall,using this culture system,isolated human OPCs not only can be stably cultured and proliferated in vitro,but also have the capacity to differentiate into oligodendrocytes.From this reproducible method,a large number of human OPCs can be stably obtained in vitro as convenient as possible.And this will provide a new option for clinical applications.This method uses fewer cytokines.Therefore,this method will provide stable,efficient and economical OPCs for cell therapy of myelination disorders or myelin damage diseases.
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Injured primary sensory axons fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Why axons stop or turn around at the DREZ has generally been attributed to growth-repellent molecules associated with astrocytes and oligodendrocytes/myelin. The available evidence challenges the contention that these inhibitory molecules are the critical determinant of regeneration failure. Recent imaging studies that directly monitored axons arriving at the DREZ in living animals raise the intriguing possibility that axons stop primarily because they are stabilized by forming presynaptic terminals on non-neuronal cells that are neither astrocytes nor oligodendrocytes. These observations revitalized the idea raised many years ago but virtually forgotten, that axons stop by forming synapses at the DREZ.
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Animals , Astrocytes , Axons , Chronic Pain , Oligodendroglia , Presynaptic Terminals , Regeneration , Spinal Cord , Spinal Nerve Roots , SynapsesABSTRACT
Objective: To observe the proliferation, migration and differentiation ofhighly enriched oligodendrocyte precursor cells (OPCs) in vitro. Methods: OPCs were dissociated and purified by shaking method from cultured glia mixture, which was derived from postnatal 1 day SD rats. BrdU incorporation assay was applied to analyze the proliferation ability of OPCs. Trans well and reaggragration assay were employed to determine the migration ability of OPCs. In differentiation model, the maturation ability of OPCswas examined by immunostaining and Western blotting analysis of myelin basic protein (MBP). Results: The stratification of glia mixture occurred 10 days after culture. The microglia was removed by first session of shaking, and there were multiple aggregated OPCs scattered on the surface of astrocyte layer. Theseparated OPCs were positive for NG2 and A2B5. Ara-C greatly inhibited the proliferation of OPCs as observed by BrdU incorporation assay. PDGF obviously promoted migration of OPCs in both Transwell assay and reaggregation models. The ratio of MBP positive cells (mature oligodendrocytes) was increased in the differentiation medium. Besides, Western blotting analysis showed that MBP in differentiation medium was greatly elevated compared to that in proliferation medium (P<0. 01). Conclusion: The in vitro cultured and purified OPCs can maintain their fundamental characteristics of proliferation, migration and differentiation.
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Demyelination disables saltatory conduction and leads to neural dysfunction. Remyelination is mediated by oligodendrocyte precursor cells (OPCs), which are widely distributed in the central nervous system (CNS) of adults. A belter understanding of oligodendrocyte biology, mechanisms of myelination and maintenance of myelin sheaths, and the relationship of failed remyelination in the CNS with the number, migration, and myelinating ability of endogenous OPCs, will greatly improve the remyelination strategies in chronic inflammatory demyelinating diseases, including muitiple sclerosis.
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Objective To obtain highly purified oligodendrocyte precursor lineage cells in vitro and make identification.Methods The oligodendrocyte precursors were separated from astrocyte by orbital shaker and further purified by differential adhesion,and finally cultured in chemically defined serum-free medium,with appended neurotrophin 2(N2),platelet-derived growth factor(PDGF),basic fibroblast growth factor(bFGF).Immunofluorescence assay was applied to identify the separated cells with A2B5,O4,O1 and glial fibrillary acidic protein(GFAP) antibodies.Results Over 95% of cultured oligodendrocyte precursor cells were obtained.The oligodendrocyte progenitors were A2B5 and O4 positive,immature oligodendrocytes were O4 and O1 positive while GFAP were negative.Conclusions Separation and purification by shaking and differential adhesion and chemically defined medium are suitable and effective to obtain highly purified oligodendrocyte precursor cells.Cell output will increase notabily and rest in immature phase by appending both N2,PDGF and bFGF.
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Objective To investigate the effects of transplantation of oligodendrocyte precursor cells(OPCs) on the recovery of neural function in rats with spinal cord injury.Methods The spinal cord injury(T10) rat model was established by Allen's method of weight-drop injury.Ninety six SD rats were randomly divided into 4 groups(24 each): transplantation group,control group,injury-only group and sham-operation group.Seven days after spinal cord injury,the rats of transplantation group received OPCs transplantation,of control group were injected with equivalent saline,and of injury-only group were untreated.The effects of OPCs transplantation on neural functional recovery in spinal cord injured rats were measured by the behavioral test and assessments of motor evoked potentials(MEP) and somatosensory evoked potentials(SEP).Results Immediately after spinal cord injuries,the neural function of spinal cord of rats was markedly impaired.The results of behavioral test,MEP and SEP in injured rats were much worse than those in sham-operation group.Although the neural function in spinal cord injured rats improved in different degrees with the time,the results of behavioral test,MEP and SEP showed it was significantly better in transplantation group than that in control group.Conclusion The transplantation of OPCs may enhance the recovery of neural function of rats with spinal cord injuries.