Direct cloning of astrocytes from primary culture without previous immortalization

Abstract

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.

A new putative target for antisense gene therapy of glioma: Glycogen synthase

“…2A and B). These observations are in agreement with those of Mbarek and co-workers, 9 who demonstrated a positive correlation between glycogen content and proliferation rate of rat astrocytes. Compared to normal glial cells 13,24 C6-glioma cells exhibit higher glycogen content.…”

“…Rat C6 glioma cells were obtained from ATCC (LGC Promochem, Molseim, France) and cultured in DMEM medium, as previously described for rat and mouse astrocytes. 9,13 cDNAs, plasmids and transfection. Molecular characterization of mouse antisense cDNA targeting mouse astrocyte glycogen synthase (GS, E.C.…”

“…4,8 The putative significance of glycogen synthase activity on cell proliferation is deduced from the positive correlation of glycogen content and proliferation in cloned rat astrocytes. 9 In fact, some current front-line strategies that detect, monitor and treat cancer capitalize on the paradigm that there is a particular "energy budget" that typifies cancer cells. 10 The present study was designed to investigate the putative role(s) of glycogen synthase (GS), the key enzyme of glycogenesis, in meeting a tumor cell's "energy budget", as well as to elucidate the impact of failure to meet this "energy budget" on tumor proliferation and invasiveness.…”

Phylogenetic Fate Mapping: Theoretical and Experimental Studies Applied to the Development of Mouse Fibroblasts

“…Here we demonstrate an alternative to wholegenome amplification, in which we have cultured single fibroblasts briefly as clonal isolates for a few generations to extract enough genomic DNA to complete multiple PCR genotyping reactions. Many types of cells, including hepatocytes (Tateno and Yoshizato 1999), adipocytes (Sugihara et al 1987), astrocytes (Mbarek et al 1998), renal tubular cells (Springate and Taub 2007), and epithelial cells from colon (Follmann et al 2000), prostate (McKeehan et al 1984), and mammary glands (Soule and McGrath 1986) can grow in primary cell culture, if not immortally, then for at least a few generations. And for those cell types that may be terminally differentiated or otherwise incapable of reentry into the cell cycle, another possibility involves expression of a conditionally immortalizing gene from a DNA tumor virus, such as the SV40 T-antigen.…”

Stable transfection of cDNAs targeting specific steps of glycogen metabolism supports the existence of active gluconeogenesis in mouse cultured astrocytes

“…This 24-h period was kept constant during all experiments. The presence of GFAP in the astrocytes was determined using an immunodection kit (Sigma, Saint Quentin Fallavier, France) as previously described (Mbarek et al, 1998).…”