Differential expression in glial cells derived from chick embryo cerebral hemispheres at an advanced stage of development

Kentroti, Susan; Vernadakis, Antonia

doi:10.1002/(sici)1097-4547(19970201)47:3<322::aid-jnr10>3.0.co;2-3

Cited by 13 publications

(7 citation statements)

References 38 publications

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“…This difference may be due to transfection but, also, may be related to the early stage of embryogenesis when stem cells were still present. Indeed, starting the culture from advanced embryonic age (Kentroti and Vernadakis, 1997), many stable mature astrocytes were obtained although multipotential neural cells were present. In our case, the experimental design does not allow to know whether or not these muhipotential cells were still present in the cultured material and if these ceils are those that give clones.…”

Section: Discussionmentioning

confidence: 99%

Direct cloning of astrocytes from primary culture without previous immortalization

Mbarek

Verge

Hevor

1998

In Vitro Cell.Dev.Biol.-Animal

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In primary cultures, much evidence shows the existence of different subtypes of astrocytes that are not all identified. One methodology for studying these subtypes can be their cloning. The present investigation shows a method for a direct cloning of astrocytes without previous immortalization. Astrocytes from the cerebral cortex of newborn rats were cultured, purified by shaking, and harvested by trypsinization. One single astrocyte was plated in a small volume of a homemade cloning medium. After getting a colony, successive platings were made using larger and larger vessels, up to 60-mm-diameter petri dishes. Then, subcultures were made. The yield of the cloning was similar to that of common eukaryotic cell clonings. All along the cloning procedure, the cells were positively immunostained with anti-glial fibrillary acidic protein antibodies. Cloned cells from some batches were spindle-shaped, looking like fibroblasts. Nevertheless, they were immunostained with anti-glial fibrillary acidic protein antibodies, unlike true fibroblasts. These spindle-shaped astrocytes were compared to cells from an astrocytoma cell line that had the same shape. The growth pattern of the astrocytoma cells was different from that of the astrocytes cloned from the primary cultures. All the types of studied cells contained glycogen. On the basis of the criteria of morphology, of glial fibrillary acidic protein immunolabeling, and of glycogen synthesis, the cloned cells kept the characteristics of astrocytes. This study shows that it is perfectly possible to get clones of astrocytes from one astrocyte without previous immortalization, giving thus a convenient material for the study of astrocyte biology.

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Section: Discussionmentioning

confidence: 99%

Direct cloning of astrocytes from primary culture without previous immortalization

Mbarek

Verge

Hevor

1998

In Vitro Cell.Dev.Biol.-Animal

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“…Vascular disruption that occurs after high blood alcohol concentrations has been implicated as a potential trigger for the reactive gliosis (Goodlett et al, 1993). These results suggest that the effects of ethanol on astrocytes, and specifically on GFAP expression, depend on the levels of alcohol, duration, and timing of exposure relative to the stage of glial maturation (glial progenitor cells and proliferation or differentiation of astrocytes) (Kennedy and Mukerji, 1986a,b;Renau-Piqueras et al, 1989;Guerri et al, 1990) and levels of growth factors and hormones (Laping et al, 1994a;Kentroti and Vernadakis, 1997). In addition, the brain regional differences in glial response to ethanol with respect to the expression of GFAP may reflect either stage-dependent vulnerability of astrocytes (different brain regions perform specific developmental functions asynchronously) or heterogeneity of astrocytes within the different brain regions.…”

Section: Figmentioning

confidence: 99%

“…Thus, whereas Miller and Robertson (1993) used postnatal rats exposed to ethanol during gestation days 8-21, we have used fetuses (or astroglial cells) from rats fed with ethanol before and during gestation (Guerri et al, 1984). In addition, the presence of certain growth factors (Kentroti and Vernadakis, 1997) and/or hormones has been suggested to be involved in the transformation of radial glia into astrocytes and in the modulation of GFAP expression (Laping et al,l994a). Therefore, changes in these modulatory factors during critical periods of brain development might explain the different effects of alcohol.…”

mentioning

confidence: 99%

Ethanol Exposure Affects Glial Fibrillary Acidic Protein Gene Expression and Transcription During Rat Brain Development

Vallés¹,

Pitarch²,

Renau‐Piqueras

et al. 1997

Journal of Neurochemistry

106

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Exposure to ethanol during fetal development reduces the astroglial-specific marker glial fibrillary acidic protein (GFAP) and its mRNA levels in brains of fetal rats and in radial glia in primary culture, affecting the proliferation and differentiation of astrocytes. The objectives of this study were to evaluate the possible effect of ethanol on GFAP mRNA levels in astrocytes and to investigate the molecular mechanism(s) involved in ethanol-induced changes in GFAP expression by analyzing the GFAP transcription rate, GFAP mRNA stability, and GFAP DNA methylation. We show here that prenatal exposure to ethanol reduces significantly GFAP immunoreactivity and its mRNA levels in both astrocytes in primary culture and brains of pups from alcohol-fed mothers. Runoff experiments from nuclei of astrocytes indicate that ethanol exposure decreases GFAP transcription rate significantly and reduces GFAP mRNA stability slightly. DNA methylation analysis indicates that prenatal ethanol exposure induces a hypermethylated state of the GFAP DNA in fetal brains. Methylation-mediated repression of GFAP transcription could be a mechanism involved in ethanolinduced reduction of GFAP expression. Ethanol-induced alterations in GFAP expression and astroglial development may underlie the CNS dysfunctions observed after prenatal alcohol exposure. Key Words: Prenatal ethanol exposure-Brain development-Astrocytes in cultureGlial fibrillary acidic protein-Vimentin-mRNA-Transcription-Runoff-mRNA stability-Methylation-Fetal alcohol syndrome.

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“…Embryonic Day 8 White Leghorn chicks were dissected, and CFNs were harvested according to the Heidemann protocol [25]; glial cells were harvested from Embryonic Day 14 White Leghorn chicks according to the Kentroti protocol [26]. Neurons and glia were provided with the same pattern medium: Neurobasal TM medium (Gibco®) (without l-glutamine), supplemented with 1% 1× GlutaMax TM (Gibco®), 1% antibiotic/antimycotic (10,000 units/mL penicillin G sodium, 10,000 μg/mL streptomycin sulfate), 50 μg/mL gentamicin, 2.5 μg/mL amphotericin B (Sigma), and 2% B27 (Gibco®), to create cell-suspensions of 50 × 10 4 cells/mL.…”

Section: Methodsmentioning

confidence: 99%

Microfluidics-based laser cell-micropatterning system

Erdman¹,

Schmidt²,

Wan³

et al. 2014

Biofabrication

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The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo-relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested, and evaluated 1) a novel removable microfluidics-based cell-delivery biochip and 2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both 2D and 3D arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 μm/s in the radial plane and 50 μm/s in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 seconds, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system.

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Differential expression in glial cells derived from chick embryo cerebral hemispheres at an advanced stage of development

Cited by 13 publications

References 38 publications

Direct cloning of astrocytes from primary culture without previous immortalization

Direct cloning of astrocytes from primary culture without previous immortalization

Ethanol Exposure Affects Glial Fibrillary Acidic Protein Gene Expression and Transcription During Rat Brain Development

Microfluidics-based laser cell-micropatterning system

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