Coexpression Of GFAP Research Articles

Co-expression of intermediate filaments glial fibrillary acidic protein and cytokeratin in pituitary adenoma

PurposeTo analyze the co-expression of the intermediate filaments GFAP and cytokeratin in 326 pituitary adenomas with regard to the distribution pattern, the subtype of the adenoma and clinical prognostic data.MethodsTissue from 326 pituitary adenomas and 13 normal anterior pituitaries collected in the Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, between 2006 and 2009 was investigated by immunohistochemistry, immunofluorescence and electron microscopy.ResultsCo-expression of intermediate filaments GFAP and cytokeratin was associated with hormone expression in 62/278 cases (22%), but only found in 2/48 (4%) of null cell adenomas (p < 0.01). Simultaneous co-expression of GFAP and cytokeratin in the same cells was demonstrated in 26 out of 326 pituitary adenomas and in all 13 pituitaries. In pituitary intermediate filaments were demonstrated in a larger area of the cytoplasm than in adenoma (p < 0.01), however, overlapping expression was seen in 2.6% of the total area in both, pituitary and adenoma. Congenially, cells with overlapping expression were found near vessels and in follicles. Furthermore, adenomas with cellular co-expression of GFAP and cytokeratin were associated with a lower recurrence rate (7.7%) compared to adenomas without co-expression of intermediate filaments (17.8%).ConclusionsCellular co-expression of the intermediate filaments GFAP and cytokeratin in pituitary adenomas and the pituitary was demonstrated and shown to be associated with hormone expression and low recurrence rate. The results are discussed with regard to the biology of folliculostellate cells, neural transformation and tumor stem cells. This study may complement the understanding of pituitary adenoma biology.

Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.

SummaryDifferentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4–7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

Injury type-dependent differentiation of NG2 glia into heterogeneous astrocytes

The glial scar is comprised of a heterogeneous population of reactive astrocytes. NG2 glial cells (also known as oligodendrocyte progenitor cells or polydendrocytes) may contribute to this heterogeneity by differentiating into astrocytes in the injured CNS, but there have been conflicting reports about whether astrocytes comprise a significant portion of the NG2 cell lineage. By using genetic fate mapping after spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) in mice, the goal of this study was to confirm and extend upon previous findings, which have shown that NG2 cell plasticity varies across CNS injuries. We generated mice that express tdTomato in NG2 lineage cells and express GFP under the Aldh1l1 or Glt1 promoter so that NG2 glia-derived astrocytes can be detected by their expression of GFAP and/or GFP. We found that astrocytes comprise approximately 25% of the total NG2 cell lineage in the glial scar by 4 weeks after mid-thoracic contusive SCI, but only 9% by the peak of functional deficit after EAE. Interestingly, a subpopulation of astrocytes expressed only GFP without co-expression of GFAP, uncovering their heterogeneity and the possibility of an underestimation of NG2 glia-derived astrocytes in previous studies. Additionally, we used high performance liquid chromatography to measure the level of tamoxifen and its metabolites in the spinal cord and show that genetic labeling of NG2 glia-derived astrocytes is not an artifact of residual tamoxifen. Overall, our data demonstrate that a heterogeneous population of astrocytes are derived from NG2 glia in an injury type-dependent manner.

Neonatal seizures induced by pentylenetetrazol or kainic acid disrupt primary cilia growth on developing mouse cortical neurons

Neonatal or early-life seizures (ELS) are often associated with life-long neurophysiological, cognitive and behavioral deficits, but the underlying mechanisms contributing to these deficits remain poorly understood. Newborn, post-migratory cortical neurons sprout ciliary buds (procilia) that mature into primary cilia. Disruption of the growth or signaling capabilities of these cilia has been linked to atypical neurite outgrowth from neurons and abnormalities in neuronal circuitry. Here, we tested the hypothesis that generalized seizures induced by pentylenetetrazol (PTZ) or kainic acid (KA) during early postnatal development impair neuronal and/or glial ciliogenesis. Mice received PTZ (50 or 100mg/kg), KA (2mg/kg), or saline either once at birth (P0), or once daily from P0 to P4. Using immunohistochemistry and electron microscopy, the cilia of neurons and glia were examined at P7, P14, and P42. A total of 83 regions were analyzed, representing 13 unique neocortical and hippocampal regions. Neuronal cilia were identified by co-expression of NeuN and type 3 adenylyl cyclase (ACIII) or somatostatin receptor 3 (SSTR3), while glial cilia were identified by co-expression of GFAP, Arl13b, and gamma-tubulin. We found that PTZ exposure at either P0 or from P0 to P4 induced convulsive behavior, followed by acute and lasting effects on neuronal cilia lengths that varied depending on the cortical region, PTZ dose, injection frequency, and time post-PTZ. Both increases and decreases in neuronal cilia length were observed. No changes in the length of glial cilia were observed under any of the test conditions. Lastly, we found that a single KA seizure at P0 led to similar abnormalities in neuronal cilia lengths. Our results suggest that seizure(s) occurring during early stages of cortical development induce persistent and widespread changes in neuronal cilia length. Given the impact neuronal cilia have on neuronal differentiation, ELS-induced changes in ciliogenesis may contribute to long-term pathology and abnormal cortical function.

Morphological Heterogeneity and Phenotype Modifications during Long Term in Vitro Cultures of Six New Human Glioblastoma Cell Lines

Long term in vitro cultures of six human malignant gliomas were established to obtain permanent lines and to assess, under conditions of prolonged culture, changes in morphology and phenotype of neoplastic cells and the extent of these modifications. We analyzed expression of the following markers by immunocytochemistry: glioma-specific antigens (GE2 and CG12), fibronectin, intermediate filaments (GFAP, vimentin, neurofilaments), class I and II histocompatibility antigens (HLA-ABC and HLA-DR), growth factor and receptor (alpha TGF and EGF-receptor), proliferation-associated antigen (Ki-67). Strong and stable staining with the two antiglioma monoclonal antibodies (GE2 and CG12) was seen, with coexpression of GFAP and fibronectin in five of six cell lines (after 20 passages) and presence of vimentin and neurofilaments. HLA-DR expression was heterogeneous, with a peculiar intracellular compartmentation in four of six cell lines. Cells showed clear cytoplasmic positivity for alpha TGF and strong membrane staining for EGF-receptor. In previous studies we showed that these cell lines have increased copies of chromosome 7; therefore we speculate that an autocrine pathway of stimulation may maintain the neoplastic growth. The percentage of Ki-67 positive proliferating cells ranged from 40 to greater than 60%, depending on cell line and passage. A slight decrease in the positivity of some markers (GFAP, vimentin and HLA-DR in 2/6 cell lines) was observed after prolonged in vitro culture (greater than 12 months), but morphophenotypic modifications, established within a few passages after explanation, were maintained with time. A clonogenic assay showed values of plating efficiency (PE) higher than corresponding values of other similar cell lines with a tendency to increase in the late passages. PE and Ki-67 positivity were not associated with tumorigenicity into nude mice (except the Hu 197 cell line). These results indicate that, in culture, all six cell lines acquired stable morphology, a well defined antigenic phenotype and high growth rate. Further studies will be performed on these permanent cell lines to clarify differentiation steps of malignant gliomas.