Expression and function of myelin-associated proteins and their common receptor NgR on oligodendrocyte progenitor cells.

Nogo-A, oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG) are known as myelin-associated proteins that inhibit axon growth by binding a common receptor, the Nogo66 receptor (NgR). In the CNS, Nogo-A, OMgp and MAG are predominantly expressed by oligodendrocytes. As our previous study revealed that oligodendrocyte progenitor cells (OPCs) did not inhibit neurite outgrowth, it is not clear whether these myelin-associated proteins are expressed in OPCs, and what functions they perform if they are expressed in OPCs. In the present study, with OPCs induced from neural precursor cells (NPCs) derived from rat embryonic spinal cord, and oligodendrocytes differentiated from OPCs, we have observed the expression patterns of Nogo-A, OMgp, MAG and NgR in NPCs, OPCs and oligodendrocytes by immunostaining and western blot assay. We found that Nogo-A could be detected in all tested cells; OMgp could be detected in OPCs and oligodendrocytes, but not in NPCs; MAG was only detected in oligodendrocytes; while NgR could be detected in NPCs and OPCs, but not in oligodendrocytes. These results indicated that the expression pattern of MAG and NgR in OPCs was totally different from that of oligodendrocytes, which might be one of the factors that led to the discrepancy between the two cells in promoting neurite outgrowth. By respectively blocking Nogo-A, OMgp and NgR expressed on OPCs with their corresponding antibodies, we further investigated their roles in the proliferation and differentiation of OPCs, as well as the possible signal pathways involved in. Our results showed that when OPCs were cultured under proliferation condition, blocking Nogo-A, OMgp or NgR did not affect the proliferation of OPCs, but could all significantly prolong their processes. And this effect on OPC processes might involve the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. When OPCs were cultured under differentiation condition (containing tri-iodothyronine, T3), blocking Nogo-A, OMgp or NgR could all inhibit the differentiation of OPCs, and this effect might involve the extracellular signal-regulated kinases1/2 (Erk1/2) signaling pathway. These results suggested that under proliferation environment, the functions of Nogo-A, OMgp and NgR expressed in OPCs might be to control the length of processes, thus maintaining the morphology of OPCs. While in differentiation environment, the functions of Nogo-A, OMgp and NgR expressed in OPCs turned to promote the differentiation of OPCs, thus facilitating the maturation of oligodendrocytes. And NgR, as the common receptor for Nogo-A and OMgp, might be the main molecule that mediated these functions in OPCs.

Differentiation of neural precursor cell-derived oligodendrocyte progenitor cells following transplantation into normal and injured spinal cords.

Demyelination contributes to the functional deficits after spinal cord injury (SCI). Therefore, remyelination may be an important strategy to facilitate repair after SCI. Oligodendrocyte precursor cells (OPCs) are immature oligodendrocytes and can differentiate into myelin-forming cells of central nervous system under certain conditions. OPC transplantation is an attractive approach for the treatment of demyelinating diseases. In this study, we transplanted OPCs expressing green fluorescent protein (GFP-OPCs) into normal and injured rat spinal cords to evaluate the differentiation of transplanted OPCs in vivo. Unfortunately, the grafted GFP-OPCs, in spinal cord whether normal or injured, were all differentiated into astrocytes, but not oligodendrocytes. Our further study indicated that inflammatory environment might not be the key factor influencing the differentiation of OPCs. Some spinal cord components, such as bone morphogenetic proteins (BMPs), were the major factors that induced OPCs to differentiate into astrocytes. The three types of BMP receptor (BMPRIA, IB and II) could all be detected in OPCs, and the astroglial differentiation of OPCs induced by spinal cord homogenate extract (SCHE) in vitro could be blocked partly by noggin, an antagonist of BMP. These results suggested that the BMPR signal transduction pathway might be one of the key factors which determine the differentiation direction of engrafted OPCs in spinal cord.

Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway.

BACKGROUND: Neural precursor cells (NPCs) are defined by their ability to proliferate, self-renew, and retain the potential to differentiate into neurons and glia. Deciphering the factors that regulate their behaviors will greatly aid in their use as potential therapeutic agents or targets. Chondroitin sulfate proteoglycans (CSPGs) are prominent components of the extracellular matrix (ECM) in the central nervous system (CNS) and are assumed to play important roles in controlling neuronal differentiation and development. RESULTS: In the present study, we demonstrated that CSPGs were constitutively expressed on the NPCs isolated from the E16 rat embryonic brain. When chondroitinase ABC was used to abolish the function of endogenous CSPGs on NPCs, it induced a series of biological responses including the proliferation, differentiation and migration of NPCs, indicating that CSPGs may play a critical role in NPC development and differentiation. Finally, we provided evidence suggesting that integrin signaling pathway may be involved in the effects of CSPGs on NPCs. CONCLUSION: The present study investigating the influence and mechanisms of CSPGs on the differentiation and migration of NPCs should help us to understand the basic biology of NPCs during CNS development and provide new insights into developing new strategies for the treatment of the neurological disorders in the CNS.

Proliferation and differentiation of oligodendrocyte progenitor cells induced from rat embryonic neural precursor cells followed by flow cytometry.

Previous studies have shown that a cell-intrinsic timer might determine when oligodendrocyte progenitor cells (OPCs) isolated from the central nervous system (CNS) stop dividing and initiate differentiation in a defined environment. In this report, the proliferation and differentiation of OPCs induced from neural precursor cells (NPCs) were analyzed by flow cytometry combined with carboxyfluorescein diacetate succinimidyl ester labeling and propidium iodide staining, respectively. When OPCs were cultured in OPC-medium, more than 30% of cells were in S- and G2/M-phases, and continuously self-renewed without differentiation. After exposure to thyroid hormone, there was an obvious decrease in the fraction of cells in both S- and G2/M-phases (<10%). Furthermore, the OPCs no longer proliferated, but differentiated into oligodendrocytes. The dynamic proliferation and differentiation characteristics of OPCs induced from NPCs and analyzed by flow cytometry were similar to those of OPCs isolated from the CNS and analyzed by other methods. These studies indicated that the proliferation and differentiation of OPCs can be followed simply and rapidly by flow cytometry.

Panaxydol and panaxynol protect cultured cortical neurons against Abeta25-35-induced toxicity.

Amyloid beta protein (Abeta), the central constituent of senile plaques in Alzheimer's disease (AD), is known to exert toxic effects on cultured neurons. In the present study, the protective effect of panaxydol (PND) and panaxynol (PNN) on Abeta25-35-induced neuronal apoptosis and potential mechanisms were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN prior to 10 microM Abeta25-35 exposure resulted significantly in elevation of cell survival determined by MTT assay, TUNEL/Hoechst staining and western blot. Furthermore, a marked increase in calcium influx and intracellular free radical generation was found after Abeta25-35 exposure, which could be almost completely reversed by pretreatment of PND or PNN. PND and PNN could also alleviate Abeta25-35-induced early-stage neuronal degeneration. These results indicated that inhibition of calcium influx and free radical generation is a mechanism of the anti-apoptotic action of PND and PNN. Since Abeta plays critical roles in the pathogenesis of AD, these findings raise the possibility that PND and PNN reduce neurodegeneration in AD.

Expression and regulation of major histocompatibility complex on neural stem cells and their lineages.

The expression of major histocompatibility complex (MHC) antigens on neural stem cells (NSCs) and their lineages is tightly related to the fate of these cells as grafts in allogenic transplantation. In this study, we observed that NSCs derived from embryonic rat forebrain expressed MHC class I and class II molecules at a low level, whereas the cells differentiated from NSCs, including neurons, astrocytes, and oligodendrocytes, lost their MHC expression. However, a proinflammatory factor, interferon-gamma (IFN-gamma), could induce and up-regulate the expression of MHC in both NSCs and their differentiated lineages in vitro. These results suggest that predifferentiating NSCs into lineage-limited cells prior to transplantation combined with controlling the local production of proinflammatory cytokines moderately may potentially benefit the survival of transplants.

Expression and regulation of versican in neural precursor cells and their lineages.

AIM: To have a better understanding of the expression and regulation of versican isoforms in neural precursor cells (NPC) and oligodendrogliogenesis. METHODS: By immunocytochemistry, RT-PCR, and real-time PCR, we examined the temporal expression of versican in NPC isolated from embryonic d 16 rats as well as in oligodendrocyte (OL) lineage cells induced to differentiate from NPC, which mimicked the oligodendrogliogenesis in vivo. RESULTS: We found that versican was constitutively expressed in NPC and their lineage cells, including neurons, astrocytes, and OL. In addition, 2 versican isoforms, V1/V0 and V2, were found to express at low levels in NPC, but at significantly higher levels in OL lineage cells. The peak expression of versican V2 was found at the oligodendrocyte precursor cell stage. Furthermore, the treatment of 2 pro-inflammatory cytokines, TNF-alpha and IFN-gamma, enhanced the transcription of versican V2 in NPC in a dose-dependent manner, but showed no effect on V1/V0 expression. CONCLUSION: Taken together, our results demonstrate that versican, particularly the inhibitory V2 isoform, is increasingly expressed in OL lineage cells induced to differentiate from NPC. An increase in versican V2 expression after cytokine stimulation implies the interplay between the injury-induced upregulation of inflammatory cytokines and chondroitin sulfate proteoglycan-mediated inhibition of axonal regeneration after central nervous system injury.

A simplified method for generating oligodendrocyte progenitor cells from neural precursor cells isolated from the E16 rat spinal cord.

Conditioned medium obtained from B104 neuroblastoma cells (B104CM) has been used widely for inducing oligodendrocyte progenitor cells (OPCs) from neural precursor cells (NPCs). Our previous studies have demonstrated that E16 rat spinal cord-derived NPCs could be induced to differentiate into OPCs using a combination of B104CM and basic fibroblast growth factor (bFGF). Here we report the development of a more efficient and reliable approach to generate large quantities of highly purified OPCs from spinal cord-derived NPCs using a combination of platelet derived growth factor (PDGF) and bFGF. We demonstrated that, after the two factors application, over 90% cells displayed typical bipolar or tripolar morphology and expressed markers for OPCs including A2B5 (90.36 +/- 4.59%), NG2 (93.63 +/- 3.37%) and platelet derived growth factor alpha receptor (PDGFR; 90.35 +/- 1.95%). Our results indicated that the PDGF/bFGF combination is more efficient in generating OPCs than the B104CM/bFGF. And it is a more potent combination of factors in promoting proliferation of OPCs.

Methods for isolating highly-enriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations.

Spinal cord neuronal culture is a useful system to study normal and abnormal functions of the spinal cord. For many bioassays, obtaining large quantities of highly purified spinal cord neurons is required. However, technical difficulties exist in obtaining these cells reliably and consistently. By comparing two dissociation methods, mechanical and enzymatic dissociations, we found that the enzymatic dissociation of embryonic day 14-15 spinal cords resulted in significantly higher cell yield than the mechanical dissociation (25.40 +/- 5.41 x 10(6) versus 3.43 +/- 0.52 x 10(6) cells per 12 embryos; n = 6/group; p < 0.01). Furthermore, cell viability was significantly higher after the enzymatic than the mechanical dissociation (83.40 +/- 3.08% versus 32.81 +/- 3.49%, n = 4/group; p < 0.01). In both methods, highly purified populations of primary neurons were obtained (mechanical: 85.17 +/- 2.84%; enzymatic: 87.67 +/- 2.52%; n = 3/group). Critical measures that affect culture outcomes include, but not limited to, the age of embryo, cell seeding density, dissociation time, and elimination of non-neuronal cells. Thus, the present study has identified the enzymatic dissociation method to be a preferred method for obtaining large quantity of highly-enriched embryonic spinal cord neurons.

Protective effect of panaxydol and panaxynol on sodium nitroprusside-induced apoptosis in cortical neurons.

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. The protective effect of panaxydol (PND) and panaxynol (PNN) on sodium nitroprusside (SNP)-induced neuronal apoptosis and potential mechanism were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN for 24 h following 1mM SNP, an exogenous NO donor, exposure for 1h, resulted significantly in reduction of cell death induced by SNP determined by MTT assay, LDH release and Hoechst staining. 5 microM PND and PNN also reduced the up-regulation of the pro-apoptotic gene, Bax, down-regulation of the anti-apoptotic gene, Bcl-2. The observations demonstrated that PND and PNN protect neurons against SNP-induced apoptosis via regulating the apoptotic related genes. The results raise the possibility that PND and PNN reduce neurodegeneration in the Alzheimer's brain.

Induction of rat neural stem cells into oligodendrocyte precursor cells.

We have previously established a culture method to isolate and cultivate neural stem cells (NSCs) derived from the rat embryonic brain and spinal cord. In the present study, we demonstrate that the spinal cord-derived NSCs can be induced to differentiate into oligodendrocyte precursor cells (OPCs) with a combined treatment composed of (1) conditioned medium collected from B104 neuroblastoma cells (B104CM) and (2) basic fibroblast growth factor (bFGF, 10 ng/ml). After induction, over 95% of the cells displayed bipolar or tri-polar morphology and expressed A2B5 and platelet derived growth factor receptor-alpha (PDGFR-alpha), markers that are specific for OPCs. Among PDGFR-alpha positive OPCs, only a few cells expressed glia fibrillary acidic protein (GFAP) and none expressed beta-tubulin III. In the presence of B104CM and bFGF, OPCs proliferated rapidly, formed spheres, expanded for multiple passages, and maintained their phenotypic properties. Upon withdrawal of B104CM and bFGF, these cells differentiated into either O4/GlaC-positive oligodendrocytes (OLs) or GFAP- and A2B5-positive type-2 astrocytes. Our results indicate that NSCs can be induced to differentiate into OPCs that possess properties of self-renewal and differentiation into oligodendrocytes and type-2 astrocytes, a property similar to that of O-2A progenitor cells. The OPCs can be maintained in an undifferentiated state over multiple divisions as long as both B104CM and bFGF are present in the medium. Thus, large quantity of OPCs can be obtained through this method for potential therapeutical interventions for various neurological degenerative diseases.

Differential gene expression in neural stem cells and oligodendrocyte precursor cells: a cDNA microarray analysis.

The use of neural stem cells (NSCs) or their progeny oligodendrocyte precursor cells (OPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features during the transition from NSCs to OPCs remain unclear. In the present study, we isolated NSCs from the embryonic rat forebrain and induced them into OPCs by using B104 conditioned medium (B104CM) in vitro. We then employed cDNA array technology to compare changes in gene expression between the two cell populations. Among 1,176 genes examined, 40 were differentially expressed, and some of them may be involved in OPC differentiation from NSCs. Our findings thus provide new insights into the molecular basis of differentiation of OPCs from NSCs.

[Isolation and cultivation of neural stem cells from the embryonic rat brain and spinal cord].

The aim of this study was to establish the culture system of isolation and cultivation of the neural stem cells (NSCs) from the embryonic rat brain and spinal cord. The methods of microscopic dissection, cell culture and immunofluorescence cytochemistry were used. The results are as follows. (1) In the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF), both brain- and spinal cord-derived stem cells proliferated and expanded in vitro for 8 - 10 passages (over 60 d). The period of expansion resulted in a 10(6)-fold increase in brain-derived NSCs and 10(5)-fold increase in spinal cord-derived NSCs. These proliferating cells expressed nestin. (2) In the medium containing 1% FBS, the two NSCs populations could be induced to differentiate into neurons, astrocytes and oligodentrocytes. The percentage of neurons (beta-tubulin III-ir) differentiated from brain-derived NSCs decreased rapidly from 11.95+/-2.5% at passage 2 (P(2)) to 1.97+/-1.16% at passage 5 (P5). Significant difference was shown between P(2) and P(5) (P<0.01). The percentage of oligodentrocytes (Rip-ir) differentiated from brain-derived NSCs remained mostly unchanged from 8.66+/-2.93% at P(2) to 9.12+/-1.13% at P(5). The same differentiation patterns were found in spinal cord-derived NSCs. All these results indicate that both embryonic rat brain- and spinal cord-derived NSCs can expand and proliferate in vitro through multiple passages, and retain the capacity to differentiate into all three major types of cells in the central nervous system.

[Effects of embryonic neural stem cells and glial cell line-derived neurotrophic factor in the repair of spinal cord injury].

The ability of implanted embryonic neural stem cells (NSCs) to improve survival, migration, and functional recovery following a compression spinal cord injury (SCI) was tested in adult rats. NSCs were isolated from E14-16 rat cerebral cortex and SCI was produced by using an aneurysm clip applicator applied to the 8th thoracic spinal cord according to method of Dolan and Tator. Two weeks after the injury, NSCs (4 microl of 1 x 10(4) cells/microl) were injected into the lesion site. The grafted NSCs were noted to survive and integrate with the host spinal cord 1 month after transplantation, which was demonstrated by the presence of Hoechst 33342 (a nuclear dye) pre-labeled NSCs within and surrounding the lesion site. Some of these cells remained undifferentiated and were stained with nestin, a marker for NSCs. Transplanted NSCs migrated for at least 3 mm from the injury epicenter towards both the rostral and caudal directions. Significant reduction in the lesion area (P<0.05) and improvement in inclined plane (P<0.05) and BBB locomotor rating scale (P<0.05) were found in the cases that received implantation of NSCs, as compared with those that received vehicle injection. More importantly, when glial cell line-derived neurotrophic factor (GDNF; 1.5 microg/microl) was added to the transplants, further reduction in lesion area (P<0.01) and improvement in the function were observed in the combined treatment group as compared with the vehicle infused group. Our results suggest that intraspinal treatment with NSCs and GDNF synergistically reduced lesion size and improved functional outcome after a compressive SCI in adult rats.