EXPRESSION OF TYROSINE-HYDROXYLASE IN AN IMMORTALIZED HUMAN FETAL ASTROCYTE CELL-LINE - IN-VITRO CHARACTERIZATION AND ENGRAFTMENT INTO THE RODENT STRIATUM

The use of primary human fetal tissue in the treatment of neurodegener ative disorders, while promising, faces several difficult technical an d ethical issues. An alternative approach that would obviate these pro blems would be to use immortalized cell lines of human fetal central n ervous system origin. An immortalized human fetal astrocyte cell line (SVG) has been established (45) and herein we describe the in vitro an d in vivo characteristics of this cell line which suggest that it may be a useful vehicle for neural transplantation. The SVG cell line is v imentin, GFAP, Thy 1.1 and MHC class I positive, and negative for neur ofilament and neuron specific enolase, consistent with its glial origi n. To determine whether the cell line could be used as a drug delivery system, a cDNA expression vector for tyrosine hydroxylase was constru cted (phTH/Neo) and stably expressed in the SVG cells for over 18 mont hs as demonstrated by immunohistochemistry and Western blotting of the stable transfectants. HPLC analysis of the supernatant from these cel ls, termed SVG-TH, consistently found 4-6 pmol/ml/min of 1-dopa produc ed with the addition of BH4 to the media. Furthermore, in cocultivatio n experiments with hNT neurons, PC-12 cells and primary rat fetal mese ncephalic tissue, both the SVG and SVG-TH tells demonstrated neurotrop hic potential, suggesting that they constituitively express factors wi th neuroregenerative potential. To determine the viability of these ce lls in vivo, SVG-TH cells were grafted into the striatum of Sprague-Da wley rats and followed over time. A panel of antibodies was used to un equivocally differentiate the engrafted cells from the host parenchyma , including antibodies to: SV40 large T antigen (expressed in the SVG- TH cells), human and rat MHC class 1, vimentin, GFAP, and tyrosine hyd roxylase. While the graft was easily identified with the first week, o ver the course of a four week period of time the engrafted cells decre ased in number. Concomittantly, rat CD4 and CD8 expression in the vici nity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of a 6-hydroxy dopamine lesioned rats, rotational behavior of the rat decreased as mu ch as 80% initially, then slowly returned to baseline over the next fo ur weeks, parallelling graft rejection. Thus, the SVG-TH cells can ind uce a functional recovery in an animal model of Parkinson's disease, h owever as a xenograft, the SVG cells are recognized by the immune syst em.