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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS021652-01
Application #
3403000
Study Section
Neurology B Subcommittee 1 (NEUB)
Project Start
1984-12-01
Project End
1987-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Geneva
Department
Type
DUNS #
481076537
City
Geneva
State
Country
Switzerland
Zip Code
CH-1211
Erkman, L; Mattenberger, L; Kato, A C (1992) A monoclonal antibody distinguishes anterior horn cells of human embryonic spinal cord during a transient period of development. Brain Res Dev Brain Res 66:109-17
Erkman, L; Touzeau, G; Bertrand, D et al. (1989) Characterization of dissociated monolayer cultures of human spinal cord. Brain Res Bull 22:57-65
Erkman, L; Wuarin, L; Cadelli, D et al. (1989) Interferon induces astrocyte maturation causing an increase in cholinergic properties of cultured human spinal cord cells. Dev Biol 132:375-88
Kato, A C; Erkman, L; Touzeau, G (1987) Human spinal cord neurons in culture as a tool to study amyotrophic lateral sclerosis. Adv Exp Med Biol 209:65-70
Erkman, L; Soldati, G; James, R W et al. (1987) Partial purification of lymphoblasts after in vitro immunization increases the yield in Ig-producing hybridomas. J Immunol Methods 98:43-52