Specific characteristic of radial glia in the human fetal telencephalon

Phenotypic characteristics of cells in the developing human telencephalic wall were analyzed using electron microscopy and immunocytochemistry with various glial and neuronal cell markers. The results suggest that multiple defined cell types emerge in the neocortical proliferative zones and are differentially regulated during embryonic development. At 5-6 weeks gestation, three major cell types are observed. Most proliferating ventricular zone (VZ) cells are labeled with radial glial (RG) markers such as vimentin, glial fibrillary acidic protein (GFAP), and glutamate astrocyte-specific transporter (GLAST) antibodies. A subpopulation of these RG cells also express the neuronal markers beta III-tubulin, MAP-2, and phosphorylated neurofilament SMI-31, in addition to the stem cell marker nestin, indicating their multipotential capacity. In addition, the presence of VZ cells that immunoreact only with neuronal markers indicates the emergence of restricted neuronal progenitors. The number of multipotential progenitors in the VZ gradually decreases, whereas the number of more restricted progenitors increases systematically during the 3-month course of human corticogenesis. These results suggest that multipotential progenitors coexist with restricted neuronal progenitors and RG cells during initial corticogenesis in the human telencephalon. Since the multipotential VZ cells disappear during the major wave of neocortical neurogenesis, the RG and restricted neuronal progenitors appear to serve as the main sources of cortical neurons. Thus, the diversification of cells in human VZ and overlying subventricular zone (SVZ) begins earlier and is more pronounced than in rodents.     

A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages

Glial fibrillary acidic protein (GFAP)-positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP-positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of betaIII-tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) betaIII-tubulin- and nestin-positive cells and (ii) GFAP- and nestin-positive cells. These cells, when subsequently cultured in serum-free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down-regulated and betaIII-tubulin expression became robust. Cells of glial lineage differentiated by down-regulating nestin expression and up-regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.     

Differentiation of radial glia-like cells from embryonic stem cells

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.     

Long-lasting coexpression of nestin and glial fibrillary acidic protein in primary cultures of astroglial cells with a major participation of nestin(+)/GFAP(-) cells in cell proliferation

Nestin, a currently used marker of neural stem cells, is transiently coexpressed with glial fibrillary acidic protein (GFAP) during development and is induced in reactive astrocytes following brain injury. Nestin expression has also been found in cultures of astroglial cells, but little is known about the fate and the mitotic activity of nestin-expressing cells in this in vitro model. The present study reveals a long-lasting expression of nestin in primary cultures of astroglial cells derived from the rat brain. Over 70% of the cells were nestin(+) at 12 weeks, with a large majority coexpressing the GFAP astrocytic marker. Time-course analyses supported a transition from a nestin(+)/GFAP(-) to a nestin(+)/GFAP(+) phenotype over time, which was further increased by cell cycle arrest. Interestingly, double staining with Ki67 revealed that over 90% of cycling cells were nestin(+) whereas only 28% were GFAP(+) in a population consisting of almost equivalent numbers of nestin(+) and GFAP(+) cells. These observations indicated that nestin(+)/GFAP(-) cells are actively engaged in mitotic activity, even after 2 weeks in vitro. Part of these cells might have retained properties of neural stem cells, insofar as 10% of cells in a primary culture of glial cells were able to generate neurospheres that gave rise to both neurons and astrocytes. Further studies will be necessary to characterize fully the proliferating cells in primary cultures of glial cells, but our present results reveal a major contribution of the nestin(+)/GFAP(-) cells to the increase in the number of astrocytes, even though nestin(+)/GFAP(+) cells proliferate also.     

GFAP-positive progenitor cells produce neurons and oligodendrocytes throughout the CNS

Once thought to merely act as scaffolds in neuronal migration, recent evidence suggests that radial glia may serve as progenitors for the majority of neurons in the CNS. Cre/loxP fate-mapping experiments were carried out using a fragment of a glial-specific promoter (glial fibrillary acidic protein; GFAP) to drive expression of Cre recombinase. We show that GFAP+ progenitor cells give rise to neurons and oligodendrocytes throughout the CNS. We find very little regional heterogeneity in the neurogenic potential of radial glia between dorsal and ventral telencephalon. Additionally, radial glia serve as precursors for subpopulations of interneurons in the ventral telencephalon. Interestingly, the human GFAP promoter but not the mouse GFAP promoter is active in oligodendrocyte progenitor cells. We also demonstrate that the most commonly used Cre reporter lines are very inefficient in detecting Cre-dependent recombination in astrocytes and describe a new Cre reporter line for assessing recombination in astrocytes.     

Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and gamma-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.     

Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis

Reactive glia, including astroglia and oligodendrocyte progenitors (OPCs) are at the core of the reaction to injury in the mammalian brain with initially beneficial and later partially adverse functions such as scar formation. Given the different glial composition in the adult zebrafish brain with radial ependymoglia but no parenchymal astrocytes, we examined the glial response to an invasive stab wound injury model in the adult zebrafish telencephalon. Strikingly, already a few days after injury the wound was closed without any scar tissue. Similar to mammals, microglia cells reacted first and accumulated close to the injury site, while neither GFAP+ radial ependymoglia nor adult OPCs were recruited to the injury site. Moreover, OPCs failed to increase their proliferation after this injury, while the number of proliferating GFAP+ glia was increased until 7 days after injury. Importantly, neurogenesis was also increased after injury, generating additional neurons recruited to the parenchyma which survived for several months. Thus, these data suggest that the specific glial environment in the adult zebrafish telencephalon is not only permissive for long-term neuronal survival, but avoids scar formation. Invasive injury in the adult zebrafish telencephalon may therefore provide a useful model to untangle the molecular mechanisms involved in these beneficial glial reactions.     

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ. © 2010 Wiley‐Liss, Inc.     

MAP2 and nestin co-expression in dysembryoplastic neuroepithelial tumors

BACKGROUND: The ontogeny and maturity of neurons and oligodendroglia-like cells (OLC) found in dysembryoplastic neuroepithelial tumors (DNT) remains controversial. A developmental origin has been proposed based on the close association to cortical dysplasia and the benign microscopic and clinical course. Our goal was to characterize the expression of nestin, a neuroepithelial precursor/stem cell antigen in DNT, along with other pathological and clinical features of this entity. METHODS: The clinical and operative features of 13 surgical specimens meeting the histological criteria for DNT were reviewed. Nestin, microtubule-associated protein 2 (MAP2), neurofilament (NF) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry and confocal scanning laser microscopy. RESULTS: Select neuronal cells in all cases demonstrated strong MAP2 immunoreactivity. Nestin-positive cells of neuronal morphology were found in 6 cases. OLC demonstrated frequent selective staining for MAP2, GFAP and nestin. Confocal microscopy demonstrated numerous examples of cells co-expressing nestin and MAP2. CONCLUSIONS: Our study suggests that OLCs represent a united population of immature neuronal (nestin + MAP2) and glial (GFAP) phenotypes. Larger, morphologically recognizable neurons also showed occasional co-expression of nestin and MAP2, suggesting a degree of dysmaturity in common with their OLC counterparts. The apparent mixed lineage of OLCs lends support to theories suggesting that DNTs arise from pluripotent neuroepithelial cells.     

Nestin expression in ganglioglioma

It has been suggested that gangliogliomas represent a neoplastic transformation of a dysplastic focus or heterotopia. Other theories propose that gangliogliomas arise from multipotent stem cells with the ability to differentiate along glial and neuronal cell lines. Our goal was to characterize the expression of nestin, a neuroepithelial precursor/stem cell antigen, in gangliogliomas along with other pathological and clinical features of this entity. The clinical and operative features of 18 recent cases meeting the histological criteria for ganglioglioma were reviewed. The expression of nestin, microtubule-associated protein 2 (MAP2), neurofilament, and glial fibrillary acidic protein (GFAP) was assessed by immunohistochemistry and confocal scanning laser microscopy. Abundant MAP2- and nestin-positive neuronal cells were found by immunohistochemistry in all 18 gangliogliomas. GFAP staining was found in reactive and lesional astrocytes but not in cells of neuronal morphology. Confocal microscopy demonstrated colocalization of nestin and MAP2 in select neuronal cells. The true lineage of gangliogliomas remains controversial. Our findings confirm the presence of cells within these lesions that harbor a persistent stem cell cytoskeletal protein (nestin). Further insight into the cytoskeletal derangement of nestin-positive neuronal cells may shed further light on the pathogenesis of gangliogliomas and its associated epilepsy.     

Induction of neuronal phenotypes from NG2+ glial progenitors by inhibiting epidermal growth factor receptor in mouse spinal cord injury

Besides neural stem cells, some glial cells, such as GFAP+ cells, radial glia, and oligodendrocyte progenitor cells can produce neuronal cells. Attractively, NG2+ glial progenitors exhibit lineage plasticity, and they rapidly proliferate and differentiate in response to central nervous system (CNS) injuries. These attributes of NG2+ glial progenitors make them a promising source of neurons. However, the potential of neuronal regeneration from NG2+ glial progenitors in CNS pathologies remains to be investigated. In this study, we showed that antagonizing epidermal growth factor receptor (EGFR) function with EGFR inhibitor caused a significant number of proliferative NG2+ glial progenitors to acquire neuronal phenotypes in contusive spinal cord injury (SCI), which presumably led to an accumulation of newly generated neurons and contributed to the improved neural behavioral performance of animals. In addition, the neuronal differentiation of glial progenitors induced by EGFR inhibitor was further confirmed with two different cell lines either in vitro or through ex vivo transplantation experiment. The inhibition of EGFR signaling pathway under the gliogenic conditions could induce these cells to acquire neuronal phenotypes. Furthermore, we find that the Ras‐ERK axis played a key role in neuronal differentiation of NG2+ glial progenitors upon EGFR inhibition. Taken together, our studies suggest that the EGFR inhibitor could promote neurogenesis post SCI, mainly from the NG2+ glial progenitors. These findings support the possibility of evoking endogenous neuronal replacement from NG2+ glial progenitors and suggest that EGFR inhibition may be beneficial to CNS trauma. © 2012 Wiley Periodicals, Inc.     

碱性成纤维生长因子等诱导间质干细胞分化为神经元样细胞的研究

目的　体外定向诱导成人骨髓间质干细胞 (MSC)分化为神经元样细胞。方法　采用Ficoll Paque液 (10 77g/L)离心分离成人MSC ,体外扩增 ,分别采用含碱性成纤维细胞生长因子 (bFGF)和叔丁对甲氧酚 (BHA)或硫代甘油等试剂的无血清DMEM诱导MSC分化为神经元。免疫组化鉴定神经元烯醇化酶 (NSE)、神经丝蛋白 (NF)、胶质纤维酸性蛋白 (GFAP)、巢蛋白 (nestin)的表达。结果　成人骨髓间质干细胞在体外扩增原代可获得 5× 10 5,10代可获得 2× 10 10 个细胞。加入bFGF和BHA等诱导剂或硫代甘油诱导后 ,MSC胞体收缩 ,突起伸出 ;免疫组化显示诱导出的神经元样细胞NSE、NF、nestin表达阳性 ,GFAP阴性。结论　成人骨髓间质干细胞在体外可以分化为神经元样细胞。     

Analysis of the temporal expression of nestin in human fetal brain derived neuronal and glial progenitor cells

Nestin expression in the developing human brain was examined with the use of unique human specific anti-nestin antibodies. Double immunostaining of cell cultures and tissue sections derived from first and second trimester human fetal brain (HFB) examined the co-expression of nestin with other cell type specific phenotypic markers. The immunocytochemical analysis shows that from first to second trimester, the majority of developing glial cells exhibited a transitional state marked by co-expression of nestin and GFAP. However, the corresponding transitional state for developing neuronal cells, co-expressing nestin and MAP-2, was rarely detected. These results imply different temporal patterns of nestin expression in cells of glial and neuronal lineages. Confocal microscopy of HFB tissue section staining also revealed a similar pattern of nestin co-expression with glial and neuronal markers. Our results suggest that nestin expression alone may not identify an undifferentiated stem cell, and that progenitor cells in glial and neuronal lineages express nestin in different temporal patterns.     

胚胎大鼠嗅神经干细胞的培养及分化特性

目的建立胚胎大鼠嗅神经干细胞(NSCs)体外培养方法,研究其增殖和分化特性.方法采用添加丝裂原的无血清培养基分离、培养胚胎14 d(E14)大鼠嗅球NSCs,应用免疫细胞化学方法鉴定培养的NSCs及自然分化为特异性神经细胞的类型,测定NSCs的生长曲线.结果从E14大鼠嗅球分离、培养出表达nestin,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSCs.嗅NSCs的增殖依赖表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF),其中EGF的促分裂增殖作用明显优于bFGF.结论从E14大鼠嗅球培养出具有自我增殖和多向分化潜能的NSCs.     

神经干细胞移植治疗大鼠脑缺血再灌注损伤实验研究

目的探讨大鼠胚胎神经干细胞移植治疗局灶性脑缺血再灌注损伤的可行性。方法孕龄8~10d的大鼠神经干细胞在体外扩增后,用免疫组织化学方法分别检测神经干细胞及其分化后代的特异性标志蛋白nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达。分别于缺血后不同时间窗将神经干细胞移植到局灶性脑缺血大鼠模型的缺血半暗带和梗塞中心,移植4w后比较不同移植部位神经干细胞存活、增殖和迁移的差异。结果从胎鼠中成功培养出悬浮生长的可表达nestin的神经球,其在含血清条件下可分化为表达GFAP的胶质细胞和表达NSE的神经元。神经干细胞移植4w后可见所有移植动物的细胞都存活,梗塞中心移植的细胞存活、增殖水平明显低于半暗带移植的细胞。结论大鼠胚胎神经干细胞移植到局灶性脑缺血再灌注损伤大鼠梗塞中心和半暗带均可长期存活,其增殖能力与移植部位密切相关。     

Expression of neural stem cell surface marker CD133 in balloon cells of human focal cortical dysplasia

PURPOSE: Focal cortical dysplasia (CD) is characterized by the presence of dysmorphic neurons, laminar and columnar disorganization. A few patients with CD have balloon cells intermixed with dysmorphic neurons. The cellular characteristics of balloon cells remain unknown. This study was intended to determine further the cellular characteristics of balloon cells. METHODS: Neocortical tissue resected from five patients with medically intractable focal epilepsy due to CD was studied. The presence of balloon cells (large opalescent cells with eccentric nuclei) was confirmed in all five patients by using cresylecht violet staining. Immunocytochemistry used antibodies against markers of pluripotential stem cells (CD133), multipotential progenitor cells (nestin), antiapoptotic gene products (Bcl-2), immature neurons (beta-tubulin 3, TUJ1), immature glia (vimentin), mature neurons (MAP2 and NeuN), and astrocytes (glial fibrillary acidic protein; GFAP). RESULTS: Balloon cells (BCs) were found to be immunoreactive to Bcl-2 (46%), vimentin (41%), Nestin (28%), CD133 (28%), MAP2 (27%), GFAP (14%), and TUJ1 (10%). An extremely small number of BCs were immunopositive for NeuN. Confocal double labeling showed that balloon cells were dually immunopositive for CD133/nestin; CD133/GFAP; CD133/Bcl-2, and nestin/GFAP. CONCLUSIONS: These results show that balloon cells are heterogeneous cell populations expressing cell-surface markers for pluripotential stem cells and proteins for multipotent progenitors, or immature neurons/glia. The presence of stem cell/progenitor markers in the balloon cells could be due to a persistent postnatal neurogenesis or early embryonic insult that resulted in arrest of proliferation/differentiation at their early stages. Additionally, the coexpression of Bcl-2 in CD133-positive balloon cells suggests that a resistance to programmed cell death may be involved in the pathogenesis of cortical dysplasia.     

胚胎小鼠脊髓源性与纹状体源性神经干细胞的培养及分化特点

目的 探讨小鼠脊髓源性神经干细胞与纹状体源性神经干细胞的分离培养方法 及增殖特点,比较两种来源的神经干细胞发育时期上的异同,寻找更有利于脊髓损伤修复的种子细胞.方法 利用显微解剖、无血清培养和单细胞克隆技术在孕14 d小鼠的胎鼠的脊髓及纹状体中分离培养具有单细胞克隆能力的细胞,免疫荧光染色检测克隆细胞的神经巢蛋白(nestin)抗原和诱导分化后特异性成熟神经细胞抗原的表达,并比较两种来源的干细胞在培养及分化方向上的异同点.结果从胎鼠的脊髓和纹状体中成功分离出神经干细胞.两种来源的干细胞均具有连续克隆能力可传代培养,表达nestin.脊髓血清诱导分化后脊髓源性神经干细胞β-tubulinⅢ阳性细胞(13.5±0.8)较纹状体源性神经干细胞(17.4±1.1)减少,而nestin、GFAP阳性细胞明显增多(45.7±0.3vs 39.2±1.2;25.2±1.3 vs 18.8±0.9),差异均有统计学意义(P<0.05). 结论 依据细胞增殖特点和分化结果的区别,证实纹状体源性神经干细胞更适合用于移植修复脊髓损伤.