In vitro development of dissociated and immunomagnetically-purified embryonic chick optic tectum cells

Immunomagnetic cell separation techniques were used to purify embryonic chick optic tectum cells from 2 different developmental ages for in vitro development studies. This negative cell selection method was based on reactivity of monoclonal antibody A2B5 with cell surfaces. Purified A2B5({dollar}-{dollar}) cells obtained initially were {dollar}>{dollar}99.99% pure. Surprisingly, A2B5(+) cells rapidly appeared in the purified surface A2B5({dollar}-{dollar}) cells in direct response to the immunomagnetic depletion. After 1 day in culture, levels of A2B5(+) cells in purified cultures equalled unpurified levels ({dollar}approx{dollar}50%). Similarly, visual densities of A2B5(+) neurons were equal in purified and unpurified long-term monolayer cultures.; Degeneration of purified cells on the neuron-selective substratum polyornithine suggested that these cells contained a paucity of neurons initially after separation. Immunohistochemistry combined with {dollar}sp3{dollar}H-thymidine autoradiography of cells and monolayers demonstrated that new DNA synthesis was required for neither the acquisition of surface A2B5-antigen, nor for differentiation into neurons. These results suggest that in early embryonic vertebrate brain (days 7 and 8) cells are present which are capable of replacing depleted neurons in vitro.; With day 12 and 13 cells, nearly all purified A2B5({dollar}-{dollar}) cells were identifiable as glia by reacting with antibodies against either glutamine synthetase or galactocerebroside. Most ({dollar}approx{dollar}80%) of the purified A2B5({dollar}-{dollar}) cells in culture for one day became A2B5(+). No increase in the percentage of A2B5(+) cells from 45% was observed in unpurified cultures. Long-term monolayer cultures from purified cells contained many A2B5(+) cells with a flattened glial or round morphology. These A2B5(+) cells frequently reacted with antibodies against glial fibrillary acidic protein and another glial marker, 5A11, which indicated a partial glial character. Additionally, flattened glial-like cells were found to contain elaborate networks of anti-neurofilament-M reactive filaments. The above combinations of markers were not found in unpurified monolayers and are believed to be a result of the immunomagnetic removal of neurons. It is hypothesized that the abnormal phenotypes in purified cell cultures from day 12 and 13 cells represent unsuccessful responses of the glia to replenish depleted neurons most likely due to restricted developmental potentials.