Fetal human brain cells were separated from 16 brain tissue samples collected after elective pregnancy termination over a Percoll density gradient, collected in two fractions, and analyzed by flow cytometry.
Immature cells were identified using anti-PSA-NCAM, anti-nestin antibodies, and the monoclonal A2B5 antibody. Oligodendrocytes were identified with the O4 and O1 monoclonal antibodies, while neurons were labeled with anti–tubulin-bIII and anti–MAP-2ab antibodies. HAM56 antibody was used to detect microglia. Anti-GFAP antibodies were used to identify astrocytes.
Human brain tissue was collected after elective termination of intrauterine pregnancies performed between gestational weeks 16 and 21. Brain tissue was rinsed several times with HBSS containing 5 mM HEPES without calcium, magnesium, or phenol red (HBSS w/o). Meninges and any residual debris were then removed. The tissue was mechanically minced into 1 mm3 or smaller pieces by a razor, rinsed with HBSS w/o, and dissociated by trypsin (0.25%), at 37°C for 45 min with shaking. The enzymatic activity of trypsin was quenched with FBS (2 ml) and the solution was treated with of DNase-I, grade II (0.05 mg/ml, approximately 2,000 U/mg) for 5 min. The tissue was spun at low speed (500 rpm), and the supernatant was discarded. DNase-I (1mg) was added to the pellet, and the tissue was triturated 20–30 times with a10-ml serologic pipette. The resultant suspension was centrifuged to form a cellular pellet. The cells were resuspended, filtered through 20-μm nylon mesh and placed in a 42-ml Oak Ridge centrifuge tube containing Percoll (12 ml) and DNase-I (1 mg). The volume was adjusted with HBSS w/o to reach 28.5% Percoll. Two cell fractions above the eythrocytes were separately collected and centrifuged. The supernatant was discarded, and the cells were resuspended in culture medium.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
