title: In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions.
abstract: Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron-glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for beta-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10-11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15-18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that > 62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical Center, Worcester, USA.
authors.forename: R
authors.initials: R
authors.lastname: Hulspas
authors.forename: C
authors.initials: C
authors.lastname: Tiarks
authors.forename: J
authors.initials: J
authors.lastname: Reilly
authors.forename: C C
authors.initials: CC
authors.lastname: Hsieh
authors.forename: L
authors.initials: L
authors.lastname: Recht
authors.forename: P J
authors.initials: PJ
authors.lastname: Quesenberry
mesh_terms: D000362:Agar; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D003260:Contact Inhibition / Q000187:drug effects*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D017930:Genes, Reporter; D008747:Methylcellulose; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000032:analysis; D009457:Neuroglia / Q000166:cytology*; D009474:Neurons / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free; D009419:Nerve Tissue Proteins; D004815:Epidermal Growth Factor; D000362:Agar; D008747:Methylcellulose; D001616:beta-Galactosidase