Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

Anti dlk1 antibody

Dopaminergic neurons are located in many regions in the CNS and are characterized by the expression of tyrosine hydroxylase (TH). TH catalyzes the rate-limiting step in the biosynthesis of dopamine utilizing tyrosine, molecular oxygen and tetrahydrobiopterin as co substrates in the formation of 3,4-dihydroxyphenylalanine. Dopaminergic neurons derived from the ventral midbrain are of special interest because of the selective loss of this cell population in patients with Parkinson’s disease (PD).

However, the study of this population of cells has been difficult because of the heterogeneity of cell cultures established from the midbrain. In most instances, dopaminergic neurons comprise five percent or less of the total cell population, which also complicates drug screening and gene discovery based on such cultures. The establishment of a method to purify dopaminergic neurons will be beneficial in the context of implanting developing dopaminergic neurons originating from aborted human fetuses in the brains of patients with PD (Bjorklund, Novartis Found Symp 2000; 231:7-15).

Although a successful restoration of function in the patients was observed in many cases, undesirable side-effects were observed in a recent study (Freed et al., 2001 N. Engl. J. Med. 344, 710-9). The problems in this study may be caused by the use of heterogeneous cell populations and uncertainties in number of dopaminergic cells transplanted (Dunnett; Nat Rev Neurosci 2001 May; 2 (5): 365-9).

Dopaminergic neurons can be visualized in formalin-fixed cell preparations, by immunostaining for tyrosine hydroxylase (TH). However, presently a method to quantify and isolate this specific cell population without harming the cells remains to be established. We show in the present project that antibodies to Fetal antigen 1 (FA1) can be used to isolate dopaminergic neurons.

FA1 is one of the increasing numbers of proteins belonging to the epidermal growth factor (EGF)-superfamily that have been identified within the last decade. FA1 is synthesized as a larger transmembrane precursor and released from cells after proteolytic action of an unidentified enzyme. Several groups have described cDNA clones for this precursor, each assigning a new name for the cDNA depending on the species and tissue/cell type from which they isolated it.

Expression of FA1 in the adult CNS has been observed (Harken-Jensen Ph.D. thesis; Odense Univ. 1999) and FA1 expression has been observed in the fetal CNS (Floridon et al., Differentiation 2000 66(1), 49-59) both authors gave preliminary results wherein the cellular location of expression was not determined. Together with our collaborators at Odense University (Harken-Jensen and Teisner), we have in this study demonstrated that the FA1 expression is a hallmark of dopaminergic neurons.

We show in the present study that FA1 antibody can be used to separate dopaminergic neurons or neural stem/progenitor cells from other neural cells. Generally, a cell suspension prepared from human CNS tissue (e.g. from human fetal brain) is brought into contact with a FA1 antibody and cells that bind FA1 antibody are then separated from cells not expressing FA1. The CNS tissue may be taken from any part of the brain or spinal cord and may be selected by dissection of particular regions, which contain particular cell types. For instance the ventral mesencephalon may be selected to provide dopaminergic neurons and the substantia nigra pars compacta is particularly rich in dopaminergic neurons.

Cell sorting using FA1 antibodies may be useful for isolation of a highly enriched source of dopaminergic neurons for transplantation into patients with PD. Antibodies that label the populations of neural stem cells, neural progenitor cells and their differentiated progeny are extremely useful in drug screening, gene discovery and for transplantation purposes because they allow the enrichment of populations of e.g. dopaminergic neurons or their progenitors in a single step. Cells recovered with FA1 antibody derived from different stages in their development could be used in studies on the mechanisms of action of cells, factors, and genes that regulate dopaminergic cell proliferation and differentiation. Furthermore, dopaminergic neurons from normal and pathological brain tissue may be recovered using FA1 antibodies and compared.

Contact

Lars WAHLBERG, (Vice President)
Tél.: +45-44608913
Fax: +45-44608989
E-mail