The grafting of fetal tissue in the treatment of neurodegenerative disorders would be greatly facilitated if a viable human fetal cell line could be substituted for primary fetal tissue. A permanent, immortalized human fetal astrocyte cell line (SVG) has been established which maintains the phenotypic characteristics of fetal astrocytes. The cDNA for human tyrosine hydroxylase, type 2, was stably transfected into the SVG cell line, establishing a second cell line, SVG-TH. These cells have continuously expressed TH for the past eighteen months, with no appreciable change over time. HPLC analysis of the supernatant from these cells consistently found 4-6 pmol/ml/min of L-dopa produced but only if BH4 was added to the media. Unexpectedly, the SVG-TH were also found to secrete serotonin, which was not found in the parent SVG cells. To determine the viability of these cells in vivo, SVG-TH cells were grafted into the normal striatum of Sprague-Dawley rats and followed over time. A panel of antibodies were used to unequivocally 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, serotonin and tyrosine hydroxylase. While the graft was easily identified with the first week, over the course of a four week period of time the engrafted cells decreased in number. Concomitantly, rat MHC class I, class II, CD4, and CD8 expression in the vicinity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of 6-hydroxydopamine-treated rats, rotational behavior of the rat decreased by 50% initially, then slowly returned to baseline over the next four weeks, paralleling graft rejection. In contrast, the SVG cell line has been successfully engrafted and survived in the striatum of the rhesus macaque for up to 9 months, immunologically acting as an allograft. MPTP- lesioned macaques are currently being characterized for subsequent engraftment with the SVG-TH cell line. We have also constructed mammalian expression vectors which express either wild type SV40 T protein or the first 155 amino acids of the N-terminus of SV40 T protein to determine whether sub-domains of the oncoprotein are capable of immortalizing primary cells without altering the phenotypic properties of the primary cells.
