RESUMEN
Astrocytes exclude Schwann cells (SCs) from the central nervous system (CNS) at peripheral nerve entry zones and restrict their migration after transplantation into the CNS. We have modeled the interactions between SCs, astrocytes, and fibroblasts in vitro. Astrocytes and SCs in vitro form separate territories, with sharp boundaries between them. SCs migrate poorly when placed on astrocyte monolayers, but migrate well on various other surfaces such as laminin (LN) and skin fibroblasts. Interactions between individual SCs and astrocytes result in long-lasting adhesive contacts during which the SC is unable to migrate away from the astrocyte. In contrast, SC interactions with fibroblasts are much shorter with less arrest of migration. SCs adhere strongly to astrocytes and other SCs, but less well to substrates that promote migration, such as LN and fibroblasts. SC-astrocyte and SC-SC adhesion is mediated by the calcium-dependent cell adhesion molecule N-cadherin. Inhibition of N-cadherin function by calcium withdrawal, peptides containing the classical cadherin cell adhesion recognition sequence His-Ala-Val, or antibodies directed against this sequence inhibit SC adhesion and increase SC migration on astrocytes. We suggest that N-cadherin-mediated adhesion to astrocytes inhibits the widespread migration of SCs in CNS tissue.
Asunto(s)
Astrocitos/fisiología , Cadherinas/fisiología , Células de Schwann/fisiología , Nervio Ciático/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Astrocitos/citología , Calcio/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Microscopía por Video , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Células de Schwann/citología , Piel/citologíaRESUMEN
Repair of demyelination in the CNS requires that oligodendrocyte precursors (OPs) migrate, divide and then myelinate. Repair of axon damage requires axonal regeneration. Limited remyelination and axon regeneration occurs soon after injury, but usually ceases in a few days. In vivo and in vitro experiments have shown that astrocytic environments are not very permissive for migration of OPs or for axonal re-growth. Yet remyelination and axon sprouting early after injury occurs in association with astrocytes, while later astrocytes can exclude remyelination and prevent axon regeneration. A large and changing cast of cytokines are released following CNS injury, so we investigated whether some of these alone or in combination can affect the ability of astrocytes to support migration of OPs and neuritic outgrowth. Interleukin (IL) 1alpha, tumour necrosis factor alpha, transforming growth factor (TGF) beta, basic fibroblast growth factor (bFGF), platelet-derived growth factor and epidermal growth factor alone exerted little or no effect on migration of OPs on astrocytes, whereas interferon (IFN) gamma was inhibitory. The combination of IL-1alpha + bFGF was found to be pro-migratory, and this effect could be neutralized by TGFbeta. We also examined neuritic outgrowth from dorsal root ganglion explants in three-dimensional astrocyte cultures treated with cytokines and found that IL-1alpha + bFGF greatly increased axon outgrowth and that this effect could be blocked by TGFbeta and IFNgamma. All these effects were absent or much smaller when OP migration or axon growth was tested on laminin, so the main effect of the cytokines was via astrocytes. The cytokine effects did not correlate with expression on astrocytes of laminin, fibronectin, tenascin, chondroitin sulphate proteoglycan, N-cadherin, polysialyated NCAM (PSA-NCAM), tissue plasminogen activator (tPA) or urokinase (uPA).
Asunto(s)
Astrocitos/efectos de los fármacos , Axones/fisiología , Citocinas/farmacología , Oligodendroglía/fisiología , Células Madre/fisiología , Animales , Astrocitos/fisiología , Recuento de Células/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Combinación de Medicamentos , Proteínas de la Matriz Extracelular/metabolismo , Sustancias de Crecimiento/farmacología , Laminina/farmacología , Mitomicina/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Activadores Plasminogénicos/metabolismo , RatasRESUMEN
The ability of cells to migrate through tissues depends on their production of a variety of proteases, and the same may be true of growth cones. Urokinase (plasminogen activator) regulates much of the extracellular proteolytic activity, by activating other proteases and as a result of its own proteolytic activity. In order to evaluate the potential role of urokinase as a promoter of axon growth, we have used a plasmid expressing urokinase under a cytomegalovirus promoter to transfect an astrocyte cell line, Neu7, which we have previously shown to provide a poor environment for axon regeneration. Five transfected lines all showed greatly increased ability to promote axon regeneration in both monolayer and three-dimensional cultures. The critical change in the transfected cells was largely within the extracellular matrix, since extracellular matrix laid down by urokinase-secreting cells was more permissive to axon growth than matrix from the parent Neu7 line. The effect was due to urokinase since treatment of the transfected cells with the urokinase inhibitors B623 and B428 rendered both the cells and their matrix much less permissive to axon growth, but did not require plasminogen, since it was blocked neither by serum-free medium nor by plasmin inhibitors.
Asunto(s)
Astrocitos/fisiología , Axones/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Animales , Astrocitos/citología , Astrocitos/ultraestructura , Axones/ultraestructura , Línea Celular , Medio de Cultivo Libre de Suero , Matriz Extracelular/fisiología , Ganglios Espinales/fisiología , Ganglios Espinales/ultraestructura , Ratones , Regeneración Nerviosa , Neuronas/fisiología , Neuronas/ultraestructura , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
Oligodendrocytes populate developing white matter and repopulate demyelinated regions of the CNS by migration. Although little is known about their migratory routes, the environment through which these cells migrate, whether during development, disease, or injury, is packed with astrocytes infiltrated with or bounded by meningeal cells. In the present study, the migration of oligodendrocyte precursors from primary cultures and of the precursor cell lines (CG4 and Oli-neu) on astrocytes and meningeal cells was investigated using tissue culture migration assays and time lapse video microscopy. Oligodendrocyte precursors and the cell lines were found to migrate poorly on astrocytes and meningeal cells compared to migration on laminin even though both astrocytes and meningeal cells express cell surface laminin. The migration-inhibitory activity was not detected in conditioned media derived from either astrocytes or meningeal cells, nor was it detected from matrix deposited by these cells. Analyses of the events immediately following cell-cell contacts revealed that oligodendrocyte precursor-astrocyte contacts were typically long-lasting and appeared to be adhesive, whereas precursor-meningeal cell contacts usually resulted in rapid withdrawal of the precursor cell process. No correlation was found, however, between general adhesiveness and the rate of migration. Our results suggest that both astrocytes and meningeal cells retard migration of oligodendrocyte precursors, consistent with the view that they may impede the movement of oligodendrocyte precursors into CNS lesion sites.
Asunto(s)
Astrocitos/citología , Movimiento Celular , Meninges/citología , Oligodendroglía/citología , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Adhesión Celular , Células Cultivadas , Medios de Cultivo Condicionados , Ratas , Células MadreRESUMEN
The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.
Asunto(s)
Astrocitos/fisiología , Axones/fisiología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/ultraestructura , Axones/metabolismo , Axones/ultraestructura , Western Blotting , Línea Celular , Matriz Extracelular/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Proteína Ácida Fibrilar de la Glía/biosíntesis , Microscopía Electrónica , Factores de Crecimiento Nervioso/biosíntesis , RatasRESUMEN
We have recently reported that the critical difference between astrocytic cell lines that are good or poor promoters of axon growth lies in the extracellular matrix. We demonstrated that much of this difference between matrix produced by permissive and non-permissive cell lines could be ascribed to one or more dermatan/keratan sulphate proteoglycans and that these proteoglycans are able to block the neurite-promoting effect of laminin. These proteoglycans are also produced by cultures of primary astrocytes. In the present study, we have demonstrated that treatment of both astrocytic cell lines and primary astrocytes with inhibitors of proteoglycan synthesis, beta-D-xylosides and sodium chlorate, can strongly influence the axon growth promoting properties of both matrix and whole cells. Dorsal root ganglia grown on matrix or in conditioned medium from cultures treated with beta-D-xylosides or sodium chlorate had twice as many axons and the axons grew to twice the length as in control cultures. Following treatment of Neu7 cells with proteoglycan synthesis inhibitors there was also a significant reduction in the ability of Neu7 conditioned medium to block the neurite-promoting effect of laminin. Dorsal root ganglia grown on Neu7 cells treated with sodium chlorate extended 2 to 3 times the number of axons for approximately 300 mm longer distance than on control cultures. Treatment of Neu7 cells with beta-D-xylosides, however, did not make the cells less inhibitory to axon growth. We have also examined the effects of proteoglycan synthesis inhibitors on three-dimensional primary astrocyte cultures, which closely mimic the in vivo effects of astrocytes on axon growth.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Astrocitos/fisiología , Axones/fisiología , Regeneración Nerviosa/fisiología , Proteoglicanos/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Axones/efectos de los fármacos , Línea Celular , Células Cultivadas , Cloratos/farmacología , Medios de Cultivo Condicionados , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/fisiología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Ganglios Espinales/ultraestructura , Glicósidos/farmacología , Regeneración Nerviosa/efectos de los fármacos , Polilisina , RatasRESUMEN
We have produced a number of astrocytic cell lines, some of which promote abundant neurite outgrowth, some of which are poor promoters of neurite outgrowth. The critical difference between these lines lies in the extracellular matrix, cell lines that are good promoters of axon growth producing a matrix that promotes axon growth, cell lines that are poor promoters of axon growth producing a non-permissive matrix. We were unable to find any consistent correlations between promotion of axon growth and production of proteases, protease inhibitors, N-cadherin, growth cone collapsing activity, and several extracellular matrix molecules. In the present study we have compared the least permissive of our cell lines, Neu7, with the most permissive, A7. Medium conditioned by the cell lines has the same properties as the matrix, since dorsal root ganglia (DRGs) grown in conditioned medium from the Neu7 line grow axons poorly, while DRGs grown in medium conditioned by A7 or primary astrocytes grow many long axons. Since matrix produced by all the cell lines contains large amounts of laminin, we looked to see whether the cells were producing laminin-blocking activity. Medium from the Neu7 line blocked laminin, while that from the A7 and primary astrocytes did not. However, when the conditioned media were heat-treated to remove neurite-promoting activity, they all had laminin-blocking activity: the blocking activity is heat stable. The neurite-promoting properties of the conditioned media therefore probably reflect a balance between promoting molecules and blockers.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Astrocitos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Dermatán Sulfato/fisiología , Matriz Extracelular/fisiología , Glicósido Hidrolasas , Sulfato de Queratano/fisiología , Neuritas/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Línea Celular , Condroitín Liasas/farmacología , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Matriz Extracelular/efectos de los fármacos , Ganglios Espinales/citología , Laminina/farmacología , Lumican , Ratas , beta-Galactosidasa/farmacologíaRESUMEN
In many regions of the rat central nervous system, oligodendrocytes develop from migratory A2B5+ precursor cells. In the rat spinal cord, during early embryonic development the capacity for oligodendrogenesis appears to be restricted to ventral regions of the spinal cord, while cultures of postnatal rat spinal cord contain a distinct population of A2B5+ astrocyte precursors. To determine if, as in other regions of the CNS, spinal cord A2B5+ cells give rise directly to oligodendrocytes and astrocytes, the initial distribution, and subsequent dispersion, proliferation, and differentiation of spinal cord A2B5+ cells have been examined in both explant and dissociated cell cultures. Spinal cord oligodendrocytes develop from A2B5+ cells. At E14, A2B5+ cells are restricted to ventral regions of the spinal cord and as development proceeds they become more uniformly distributed throughout the spinal cord. In explant cultures, greater than 95% of the explants that contain oligodendrocytes also contain A2B5+ cells and a proportion of mature oligodendrocytes retain detectable A2B5 immunoreactivity briefly on their surface. The maturation of spinal cord oligodendrocyte precursors occurs in a number of distinct stages characterized by the expression of O4 immunoreactivity, which first appears at E16, and GC immunoreactivity, which first appears at E18. As spinal cord oligodendrocyte precursors acquire O4 immunoreactivity they appear to lose the ability to proliferate in response to PDGF but retain the ability to proliferate in response to bFGF, suggesting that the control of proliferation of oligodendrocyte precursors is, in part, dependent on their maturational state. In the presence of high serum, spinal cord A2B5+ cells fail to develop in isolated E14 dorsal spinal cord cultures, while in ventral cultures they subsequently differentiate into A2B5+ astrocytes suggesting that A2B5+ astrocyte precursors are also initially ventrally located. Unlike oligodendrocyte differentiation, however, the differentiation of spinal cord A2B5+ cells into astrocytes is delayed in early embryonic-derived cultures compared to those from older animals. These observations suggest that local influences may regulate the timing of spinal cord A2B5+ astrocyte development, but not spinal cord oligodendrocyte development.
Asunto(s)
Oligodendroglía/fisiología , Médula Espinal/crecimiento & desarrollo , Animales , Anticuerpos/inmunología , Diferenciación Celular , Células Cultivadas , Técnicas de Cultivo , ADN/biosíntesis , Femenino , Técnica del Anticuerpo Fluorescente , Oligodendroglía/inmunología , Embarazo , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/embriología , Células MadreRESUMEN
The vertebrate spinal cord is comprised of a complex array of different populations of neurons and axon tracts. Recent studies suggest that this complex neuronal cytoarchitecture is complemented by a similarly complex glial cytoarchitecture. Cultures of neonatal rat spinal cord contain multiple different classes of astrocytes. These distinct classes of astrocytes have particular morphologies and arise from separate precursors that proliferate in response to different mitogens. It seems likely that the individual classes of astrocytes will have regional localization and will be involved in specific functions in the intact spinal cord. In contrast to the significant diversity seen among astrocytes, spinal cord oligodendrocytes in the embryonic animal appear to be a relatively homogenous population of cells that proliferates in response to known growth factors at particular stages of maturation. An important future challenge in glial cell biology is to define clearly the functional roles of individual populations of astrocytes in the developing adult, and injured spinal cord. Such information may ultimately lead to the ability to modulate astrocytic function at a cellular level during aberrant development and following injury to the adult spinal cord.
Asunto(s)
Astrocitos/citología , Neuroglía/citología , Médula Espinal/citología , Envejecimiento , Animales , Astrocitos/fisiología , Diferenciación Celular , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/análisis , Humanos , Mamíferos , Oligodendroglía/citología , Ratas , Médula Espinal/crecimiento & desarrollo , Médula Espinal/fisiología , VertebradosRESUMEN
Cultures of newborn rat spinal cord contain multiple types of astrocytes. By using a combination of cultures enriched for glial precursors and clonal analysis, we have identified a particular astrocyte precursor that gives rise to morphologically distinct classes of astrocytes. This astrocyte precursor labels with the monoclonal antibody A2B5, is highly migratory, proliferates in response to serum and platelet-derived growth factor, and differentiates into process-bearing astrocytes, many of which subsequently assume a "pancake"-shaped morphology. A2B5+ astrocyte precursors share antigenic and migratory characteristics with previously described O2A progenitor cells but differ in their response to regulatory factors, including serum and coculture with type 1 astrocytes. More importantly, these astrocyte precursors do not give rise to oligodendrocytes. In their proliferative response to serum and their capacity to differentiate into astrocytes, these glial precursors resemble type 1 astrocyte precursors from optic nerve. However, unlike type 1 astrocyte precursors, these cells are A2B5+, highly migratory, and do not give rise to fibroblast-like astrocytes. Neonatal rat spinal cord cultures contain approximately twice the number of the A2B5+ astrocyte precursors than O2A progenitor cells. By contrast, the majority of A2B5+ cells in postnatal day 7 optic nerve cultures are O2A progenitors. The presence of large numbers of A2B5+ astrocyte precursors in rat spinal cord cultures may reflect the more complex cytoarchitecture of the spinal cord compared to the optic nerve.
Asunto(s)
Astrocitos/citología , Proteínas del Tejido Nervioso/análisis , Médula Espinal/citología , Animales , Animales Recién Nacidos , Anticuerpos , Diferenciación Celular , Medios de Cultivo , Proteína Ácida Fibrilar de la Glía/análisis , Neuroglía/citología , Oligodendroglía/citología , Nervio Óptico/citología , Ratas , Ratas EndogámicasRESUMEN
The neuroepithelial cells of the mammalian neural tube are thought to give rise to all classes of differentiated neurons and macroglial cells in the adult CNS. In most cases, the regulation and timing of commitment of neuroepithelial cells to specific differentiative pathways are unknown. It has been proposed that in developing spinal cord, the macroglial cells--astrocytes and oligodendrocytes--arise either by the direct transformation of radial glial cells in the developing cord or, alternatively, by the differentiation of distinct precursor cells which migrate to presumptive white matter from the region of the central canal during development. In this study, the timing of oligodendrocyte differentiation in different levels of the spinal cord and the capacity of specific regions of the spinal cord to give rise to oligodendrocytes at various ages was tested in vitro. At embryonic day 14, all complete segments, as well as all ventral regions along the rostral-caudal axis of the spinal cord, have the capacity for oligodendrogenesis. By contrast, dorsal regions of the thoracic and lumbar spinal cord do not develop the capacity for oligodendrogenesis until later in development. The capacity of dorsal rat spinal cord to give rise to oligodendrocytes appears to be associated with the ventral-to-dorsal migration of oligodendrocyte precursors. These observations suggest that commitment to an oligodendrocyte differentiative pathway appears to occur in a distinct population of ventrally located glial precursors in the embryonic rat spinal cord.
Asunto(s)
Oligodendroglía/citología , Médula Espinal/citología , Células Madre/citología , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular , Técnicas Citológicas , Desarrollo Embrionario y Fetal , RatasRESUMEN
As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.
Asunto(s)
Sistema Nervioso Central/crecimiento & desarrollo , Regeneración Nerviosa , Envejecimiento , Animales , Diferenciación Celular , División Celular , Sistema Nervioso Central/citología , Humanos , Esclerosis Múltiple/fisiopatologíaRESUMEN
The origin of oligodendrocytes and astrocytes in the CNS is still a focus of much experimentation and controversy. We have used antibodies against ganglioside GD3 and galactocerebroside (GC) to follow the origin and development of rat cerebellar oligodendrocytes both in vitro and in vivo. The immunofluorescent identification of GC+ cells in the rat neonatal cerebellum in vivo, revealed that cells initially GD3+/GC- appeared to make the transition via GD3+/GC+ cells to GD3-/GC+ oligodendrocytes. This sequence of events closely paralleled the maturation of cerebellar oligodendrocyte precursors found in serum-free dissociated culture. In contrast, whereas both GD3+ and glial fibrillary acidic protein-positive cells were seen in serum-containing dissociated culture and also in freshly dissociated suspensions of cerebellum at postnatal days 0 to 6, such cells could not be identified in situ. Putative GD3+/GC- oligodendrocyte precursor cells arose from the deeper regions of the cerebellum at birth, perhaps initially from the superior medullary velum adjacent to the fourth ventricle, and appeared to migrate into the developing folia just prior to myelination and the acquisition of GC.
Asunto(s)
Cerebelo/crecimiento & desarrollo , Neuroglía/fisiología , Oligodendroglía/fisiología , Animales , Anticuerpos Monoclonales , Recuento de Células , Movimiento Celular , Células Cultivadas , Cerebelo/citología , Técnica del Anticuerpo Fluorescente , Galactosilceramidas/análisis , Gangliósidos/análisis , Proteína Ácida Fibrilar de la Glía/análisis , Ratas , Manejo de EspecímenesRESUMEN
Cell type-specific neural markers and indirect immunofluorescence were used to study the antigenic phenotype, development and function of astrocytes and oligodendrocytes in dissociated cell cultures of human fetal optic nerve and cerebral cortex. It was found that (1) two populations of GFAP+ astrocytes could be distinguished in optic nerve cultures using the A2B5 monoclonal antibody; (2) the proportion of A2B5+ astrocytes and of oligodendrocytes which developed in optic nerve cultures was dependent on the tissue culture medium; (3) A2B5+ cells were required for the development of galactocerebroside+ oligodendrocytes and at least some GFAP+A2B5+ astrocytes; (4) astrocytes purified from cerebral cortex were A2B5- and NCAM+; and (5) astrocytes supported the unfasciculated growth of rat CNS neurons. These results demonstrate close similarities between cultured rat and human neural cells. Such observations are likely to be of significance in future studies of the developing human nervous system and of a variety of neurological diseases.
Asunto(s)
Antígenos de Superficie/análisis , Proteína Ácida Fibrilar de la Glía/análisis , Neuroglía/fisiología , Anticuerpos Monoclonales , Moléculas de Adhesión Celular , Células Cultivadas , Corteza Cerebral/citología , Técnica del Anticuerpo Fluorescente , Histocitoquímica , Humanos , Neuroglía/análisis , Neuroglía/clasificación , Nervio Óptico/citologíaRESUMEN
The rat optic nerve contains three types of macroglial cells: type 1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes at birth (E21), and type 2 astrocytes between postnatal days 7 and 10. The oligodendrocytes and type 2 astrocytes develop from a common, bipotential O-2A progenitor cell. We show here that although O-2A progenitor cells in E17 optic nerve prematurely stop dividing and differentiate into oligodendrocytes within 2 days in culture, when cultured on a monolayer of type 1 astrocytes, they continue to proliferate; moreover, the first cells differentiate into oligodendrocytes after 4 days in vitro, which is equivalent to the time that oligodendrocytes first appear in vivo. Our findings suggest that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell that counts cell divisions that are driven by a growth factor (or factors) produced by type 1 astrocytes.
Asunto(s)
Astrocitos/fisiología , Neuroglía/citología , Oligodendroglía/citología , Células Madre/citología , Animales , Relojes Biológicos , Comunicación Celular , Recuento de Células , Diferenciación Celular , División Celular , Separación Celular , Células Cultivadas , Corteza Cerebral/citología , Nervio Óptico/citología , Nervio Óptico/embriología , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
We have examined the consequences of surface interactions with glial and nonglial cells on the in vitro growth of CNS neurons. When cerebellar or spinal cord cells were plated onto monolayers highly enriched in cortical astrocytes or sciatic nerve Schwann cells, neurons generally grew as single cells and showed relatively little tendency to aggregate. Similarly, neurites showed little tendency to fasciculate. In contrast, when plated onto fibroblast, heart muscle-fibroblast, or astrocyte-free meningeal monolayers, neurons rapidly aggregated, and neurite outgrowth was primarily in large fascicles. There were no glia detectable in the majority of aggregates or fascicles, suggesting that aggregation and fasciculation were due to interactions between neurons. Neurite outgrowth over 24 hr was also greater on astrocytes than on nonglia. Whether or not aggregation and fasciculation occurred was due to surface properties of the glial and nonglial cells. When neurons were added to astrocyte and nonglial monolayers growing in medium conditioned by a large excess of co-cultured nonglia or astrocytes, respectively, the pattern of neuronal growth was determined by the type of monolayer with which the neurons were in contact. Moreover, the initial growth of neurons on heat-killed astrocytes was indistinguishable from growth on living astrocytes. The pattern of neuronal growth on these different monolayers suggests that neurons are more adherent to glia than to other neurons but are more adherent to other neurons than to nonglia. Such an adherence hierarchy could explain the consistent finding of an apposition of neurons to glial surfaces during neuronal migration and axon outgrowth. Our findings also suggest that the interaction of axons with the non-neuronal milieu through which they grow may play an important role in regulating fasciculation, a process which has generally been treated as due primarily to axon-axon interactions.