Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization.
We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3-9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte-specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP-positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1-3%) were present in the human adult astrocyte cultures, using a panel of macrophage-specific markers. However, the monoclonal antibodies (mAbs KP1, EBM1, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/ microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non-lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay).
De Groot
C J
CJ
Graduate School Neurosciences Amsterdam, Department of Pathology, Free University Academic Hospital, The Netherlands. cja.degroot@azvu.nl
Langeveld
C H
CH
Jongenelen
C A
CA
Montagne
L
L
Van Der Valk
P
P
Dijkstra
C D
CD
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D001253:Astrocytes / Q000502:physiology* / Q000648:ultrastructure; D001921:Brain / Q000473:pathology* / Q000648:ultrastructure; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000096:biosynthesis; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000473:pathology*; D010587:Phagocytosis / Q000502:physiology; D010641:Phenotype; D013116:Spinal Cord / Q000473:pathology* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins