胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

Induction of cell death in rat brain by a gliotoxic factor from cerebrospinal fluid in multiple sclerosis.

The cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients contains a 17 kDa glycoproteic factor with gliotoxic properties in vitro. In order to study the physiopathological role of this gliotoxic factor in vivo, we have injected a partially purified preparation and appropriate controls in rat CSF and investigated whether it induces cell death in the rat central nervous system (CNS), 10 days and 3 months after injection. We used the TUNEL assay in association with specific immunohistochemistry to characterize dying cells in the gliotoxic factor- treated rat CNS. At 10 days post-injection, TUNEL-positive cells were observed in the whole rat CNS. They were particularly numerous in the choroid plexus, ependymal epithelium, cerebral white matter, cerebral vascular endothelium, arachnoid spaces and less frequent in the gray matter of brain and spinal cord. The predominant type of TUNEL-positive cells observed at 10 days post-injection was astrocytes, in white matter, gray matter, occasionnally around damaged endothelial cells in periventricular and subpial spaces. Other TUNEL-positive cells were identified as oligodendrocytes by an oligodendrocyte specific RIP immunostaining, at 10 days post-treatment with the gliotoxic factor. Interestingly, demyelination and death of oligodendrocytes were more important 3 months post-injection: TUNEL-RIP positive oligodendrocytes were generally associated with multifocal demyelinating areas. Clearly, the 17 kDa gliotoxic factor injection in rat CSF triggers demyelination and may be used as a new animal model for MS. Also, our results suggest a new possible scenario for MS pathogenesis: death of oligodendrocytes and astrocytes, stimulated by the MS gliotoxic factor causes the breakdown of the blood-brain barrier (BBB) and the demyelinating cascade.     

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells

Background aimsThe widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2⁺ populations in vitro and in vivo.MethodsTwenty-four clones from embryonic cerebral cortex-derived NG2⁺ cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2⁺ clones into the spinal cord was used to examine their lineage potential in vivo.ResultsIn vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal–glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environmentConclusionsThese results suggest that NG2⁺ cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2⁺ cell lines might provide a cell source for treating spinal cord disorders.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

Cutting edge: Multiple sclerosis-like lesions induced by effector CD8 T cells recognizing a sequestered antigen on oligodendrocytes

CD8 T cells are emerging as important players in multiple sclerosis (MS) pathogenesis, although their direct contribution to tissue damage is still debated. To assess whether autoreactive CD8 T cells can contribute to the pronounced loss of oligodendrocytes observed in MS plaques, we generated mice in which the model Ag influenza hemagglutinin is selectively expressed in oligodendrocytes. Transfer of preactivated hemagglutinin-specific CD8 T cells led to inflammatory lesions in the optic nerve, spinal cord, and brain. These lesions, associating CD8 T cell infiltration with focal loss of oligodendrocytes, demyelination, and microglia activation, were very reminiscent of active MS lesions. Thus, our study demonstrates the potential of CD8 T cells to induce oligodendrocyte lysis in vivo as a likely consequence of direct Ag-recognition. These results provide new insights with regard to CNS tissue damage mediated by CD8 T cells and for understanding the role of CD8 T cells in MS.     

NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration

The mammalian CNS contains a ubiquitous population of glial progenitors known as NG2+ cells that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although it has been reported that NG2+ cells are multipotent, their fate in health and disease remains controversial. Here, we generated PDGFαR-CreER transgenic mice and followed their fate in vivo in the developing and adult CNS. These studies revealed that NG2+ cells in the postnatal CNS generate myelinating oligodendrocytes, but not astrocytes or neurons. In regions of neurodegeneration in the spinal cord of ALS mice, NG2+ cells exhibited enhanced proliferation and accelerated differentiation into oligodendrocytes but remained committed to the oligodendrocyte lineage. These results indicate that NG2+ cells in the normal CNS are oligodendrocyte precursors with restricted lineage potential and that cell loss and gliosis are not sufficient to alter the lineage potential of these progenitors.     

BMP inhibition enhances axonal growth and functional recovery after spinal cord injury

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP-2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.     

Human Mesenchymal Stem Cells Signals Regulate Neural Stem Cell Fate

Neural stem cells (NSCs) differentiate into neurons, astrocytes and oligodendrocytes depending on their location within the central nervous system (CNS). The cellular and molecular cues mediating end-stage cell fate choices are not completely understood. The retention of multipotent NSCs in the adult CNS raises the possibility that selective recruitment of their progeny to specific lineages may facilitate repair in a spectrum of neuropathological conditions. Previous studies suggest that adult human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after a wide range of CNS insults, probably through their trophic influence. In the context of such trophic activity, here we demonstrate that hMSCs in culture provide humoral signals that selectively promote the genesis of neurons and oligodendrocytes from NSCs. Cell–cell contacts were less effective and the proportion of hMSCs that could be induced to express neural characteristics was very small. We propose that the selective promotion of neuronal and oligodendroglial fates in neural stem cell progeny is responsible for the ability of MSCs to enhance recovery after a wide range of CNS injuries. Special issue dedicated to Anthony Campagnoni.     

Identity, distribution, and development of polydendrocytes: NG2-expressing glial cells

Cells that express the NG2 proteoglycan (NG2+ cells) comprise a unique population of glial cells in the central nervous system. While there is no question that some NG2+ cells differentiate into oligodendrocytes during development, the persistence of numerous NG2+ cells in the mature CNS has raised questions about their identity, relation to other CNS cell types, and functions besides their progenitor role. NG2+ cells also express the alpha receptor for platelet-derived growth factor (PDGF αR), a receptor that mediates oligodendrocyte progenitor proliferation during development. Antigenically, NG2+ cells are distinct from fibrous and protoplasmic astrocytes, resting microglia, and mature oligodendrocytes. Therefore, we propose the term polydendrocytesto refer to all NG2-expressing glial cells in the CNS parenchyma. This distinguishes them from the classical glial cell types and identifies them as the fourth major glial population in the CNS. Recent observations suggest that polydendrocytes are complex cells that physically and functionally interact with other cell types in the CNS. Committed oligodendrocyte progenitor cells arise from restricted foci in the ventral ventricular zone in both spinal cord and brain. It remains to be clarified whether there are multiple sources of oligodendrocytes, and if so whether polydendrocytes (NG2+ cells) represent progenitor cells of all oligodendrocyte lineages. Proliferation of NG2+ cells during early development appears to be dependent on PDGF, but the regulatory mechanisms that govern NG2+ cell proliferation in the mature CNS remain unknown. Pulse-chase labeling with bromodeoxyuridine indicates that polydendrocytes that proliferate in the postnatal spinal cord differentiate into oligodendrocytes. Novel experimental approaches are being developed to further elucidate the functional properties and differentiation potential of polydendrocytes.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Role of Cell Cycle Proteins in CNS Injury

Following trauma or ischemia to the central nervous system (CNS), there is a marked increase in the expression of cell cycle-related proteins. This up-regulation is associated with apoptosis of post-mitotic cells, including neurons and oligodendrocytes, both in vitro and in vivo. Cell cycle activation also induces proliferation of astrocytes and microglia, contributing to the glial scar and microglial activation with release of inflammatory factors. Treatment with cell cycle inhibitors in CNS injury models inhibits glial scar formation and neuronal cell death, resulting in substantially decreased lesion volumes and improved behavioral recovery. Here we critically review the role of cell cycle pathways in the pathophysiology of experimental stroke, traumatic brain injury and spinal cord injury, and discuss the potential of cell cycle inhibitors as neuroprotective agents. Special issue dedicated to Dr. Moussa Youdim.     

In vitro characterization of subventricular zone isolated neural stem cells,from adult monkey and rat brain

Neural stem cells (NSCs) are multipotent, self-renewable cells who are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside at the subventricular zone (SVZ) of the adult brain permanently to guarantee a lifelong neurogenesis during neural network plasticity or undesirable injuries. Although the specious inaccessibility of adult NSCs niche hampers their in vivo identification, researchers have been seeking ways to optimize adult NSCs isolation, expansion, and differentiation, in vitro. NSCs were isolated from rhesus monkey SVZ, expanded in vitro and then characterized for NSCs-specific markers expression by immunostaining, real-time PCR, flow cytometry, and cell differentiation assessments. Moreover, cell survival as well as self-renewal capacity were evaluated by TUNEL, Live/Dead and colony assays, respectively. In the next step, to validate SVZ-NSCs identity in other species, a similar protocol was applied to isolate NSCs from adult rat’s SVZ as well. Our findings revealed that isolated SVZ-NSCs from both monkey and rat preserve proliferation capacity in at least nine passages as confirmed by Ki67 expression. Additionally, both SVZ-NSCs sources are capable of self-renewal in addition to NESTIN, SOX2, and GFAP expression. The mortality was measured meager with over 95% viability according to TUNEL and Live/Dead assay results. Eventually, the multipotency of SVZ-NSCs appraised authentic after their differentiation into neurons, astrocytes, and oligodendrocytes. In this study, we proposed a reliable method for SVZ-NSCs in vitro maintenance and identification, which, we believe is a promising cell source for therapeutic approach to recover neurological disorders and injuries condition.

     

A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.     

Cellular Environment Directs Differentiation of Human Umbilical Cord Blood�CDerived Neural Stem Cells In Vitro

Cord blood–derived neural stem cells (NSCs) are proposed as an alternative cell source to repair brain damage upon transplantation. However, there is a lack of data showing how these cells are driven to generate desired phenotypes by recipient nervous tissue. Previous research indicates that local environment provides signals driving the fate of stem cells. To investigate the impact of these local cues interaction, the authors used a model of cord blood–derived NSCs co-cultured with different rat brain–specific primary cultures, creating the neural-like microenvironment conditions in vitro. Neuronal and astro-, oligo-, and microglia cell cultures were obtained by the previously described methods. The CMFDA-labeled neural stem cells originated from, non-transformed human umbilical cord blood cell line (HUCB-NSCs) established in a laboratory. The authors show that the close vicinity of astrocytes and oligodendrocytes promotes neuronal differentiation of HUCB-NSCs, whereas postmitotic neurons induce oligodendrogliogenesis of these cells. In turn, microglia or endothelial cells do not favor any phenotypes of their neural commitment. Studies have confirmed that HUCB-NSCs can read cues from the neurogenic microenvironment, attaining features of neurons, astrocytes, or oligodendrocytes. The specific responses of neurally committed cord blood–derived cells, reported in this work, are very much similar to those described previously for NSCs derived from other “more typical” sources. This further proves their genuine neural nature. Apart from having a better insight into the neurogenesis in the adult brain, these findings might be important when predicting cord blood cell derivative behavior after their transplantation for neurological disorders.     

神经干细胞巢成分及调节机制的研究进展

神经干细胞（NSCs）是一类具有自我更新和多向分化潜能的细胞。在特定的条件下能够分化成神经元、星形胶质细胞和少突胶质细胞,从而参与神经发生和损伤修复。调节NSCs的特定微环境,通常称为神经干细胞巢,包括多个细胞群,其贡献目前正在积极探索。了解NSCs及其微环境成分之间的相互作用,对于开发治疗神经退行性疾病及脊髓损伤的疗法至关重要。本篇综述描述并讨论了最新的研究,确定了新的成分在神经干细胞巢中的作用。这些发现给这个领域带来了新的概念。本综述评估这些最新进展,提高对NSCs微环境及其对NSCs功能的影响的认识。