Characterization of fetal human brain cultures. Development of a potential model for selectively purifying human glial cells in culture.
Seven fetal human brain and three fetal human leptomeningeal cultures were characterized according to cell morphology, ultrastructural features, antigen expression, and collagen biosynthesis capabilities. Primary cultures derived from mechanically and enzymatically dissociated samples of fetal human brain consisted of a heterogeneous cell population in which astrocytes, oligodendrocytes, neurons, mesenchymal (leptomeningeal) cells, and macrophages were identified by light and electron microscopy. With progressive subcultivation, a homogeneous, leptomeningeal cell-derived population predominated. Fetal human brain and leptomeningeal specimens embedded in paraffin were analyzed immunohistochemically for the distribution of glial fibrillary acidic protein (GFAP), vimentin, factor-VIII-related antigen, fibronectin, laminin, type IV collagen, and procollagen III. Only GFAP and vimentin identified astrocytes and radial glia in the developing human brain; fibronectin, laminin, and the collagen types were immunolocalized largely to the leptomeninges and to the cerebral vasculature. The percentage of cells positively identified by antiserum to GFAP was greatest in primary cultures of fetal human brain; by the fourth passage, none of the fetal brain cultures were GFAP positive. The progressive decrease in the percentage of GFAP-positive cells was accompanied by an increase in the percentage of cells identified by collagen immunomarkers. Furthermore, in double immunolabeling experiments, antibodies to GFAP recognized a population of cells that was not identified by antibodies to laminin, fibronectin, type IV collagen, or procollagen III. SDS-PAGE and DEAE-cellulose chromatography of [3H]-proline-labeled early-passage fetal human brain cultures revealed collagen profiles identical to those obtained from direct cultures of the leptomeninges. The characteristics of later-passage fetal human brain cultures were identical in all respects to those of the fetal human leptomeningeal cultures. The proliferation of leptomeningeal cells could be inhibited by exposing the cells to cis-hydroxyproline (200 micrograms/ml). Primary fetal human brain cultures similarly treated with the proline analogue were found to be highly enriched for glial cells; these cultures were more than 90% GFAP positive. We conclude that primary fetal human brain cultures consist of a heterogeneous population of cells, most of which under the present culture conditions can be identified as glial cells. Subcultivation of human fetal brain cultures results in the overgrowth of mesenchymal cells, which are presumably derived from the leptomeninges.(ABSTRACT TRUNCATED AT 400 WORDS)
Rutka
J T
JT
Department of Neurological Surgery, School of Medicine, University of California, San Francisco.
Giblin
J R
JR
Balkissoon
R
R
Wen
D
D
Myatt
C A
CA
McCulloch
J R
JR
Rosenblum
M L
ML
D001921:Brain / Q000166:cytology / Q000196:embryology* / Q000276:immunology / Q000648:ultrastructure; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D005333:Fetus / Q000033:anatomy & histology*; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology; D006801:Humans; D006909:Hydroxyproline / Q000494:pharmacology; D007150:Immunohistochemistry; D008578:Meninges / Q000166:cytology / Q000196:embryology / Q000276:immunology / Q000648:ultrastructure; D008854:Microscopy, Electron; D009457:Neuroglia / Q000166:cytology*; D013237:Stereoisomerism
D005904:Glial Fibrillary Acidic Protein; D003094:Collagen; D006909:Hydroxyproline