We have developed an in vitro preparation, the dissociated monolayer culture of human embryonic spinal cord cells, which will be used as a tool for the study of Motor Neuron Disease (MND). For reproducible neuronal cultures, it is necessary to use embryonic tissue between the 8th and 9th week. The neurons survive as long as 7 weeks in culture and we have examined some of their morphological, biochemical and electrophysiological properties. Cholinergic neurons, characterized by choline acetyltransferase (CAT) activity and the synthesis of acetylcholine, survive and develop in these cultures. The spinal cord neurons grow in a standard culture medium which contain 13 percent control human serum; therefore human serum contains the necessary trophic factor(s) for the growth of these cells. Three hypothees for the etiology of MND will be examined using control and disease sera: (i) an anti-neuronal antibody that may be involved in the pathogenesis of the disease; this will be tested by examining the binding of immunoglobulins from MND sera on the cultured neurons, (ii) an abnormality in a trophic factor for motor neurons, and (iii) a circulating neurotoxin. The latter two hypotheses will require testing the effects of sera (fractionated and unfractionated; disease and control) on the cultured cells. After 3 weeks in culture, the neurons will be counted to determine their survival and the levels of CAT, glutamic acid decarboxylase and lactate dehydrogenase activities will be measured. Since the human spinal cord cultures consist of a heterogeneous mixture of neurons and non-neuronal cells, our ultimate goal is to isolate and culture a pure population of human embryonic motor neurons. Initially we will develop a monoclonal antibody (McAb) specific for adult human motor neurons using in vitro immunization techniques; the antigen will be a membrane fraction of large spinal neurons isolated in bulk from human autopsy spinal cord. Secondly this McAb (labelled with fluorescein) will be used as a tag for the embryonic motor neurons allowing them to be separated from the other cells of the spinal cord by means of fluorescence-activated cell sorter.
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