Fetal tissue or cell transplants as replacement therapy for neurodegenerative diseases such as Parhinson's disease (PD) and Alzheimer's disease (AD) has received considerable attention. Fetal tissue transplant has several inherent problems. These include ethical, availability, heterogeneity of cell population, and the presence of donor tissue associated antigen presenting cells which play a major role in tissue rejection. The establishment of immortalized clones of central nervous system (CNS) nerve cells, may help to alleviate the above problems. No such clones of human CNS cells are available at this time.
Our specific aims are: (a) to establish immortalized clones of fetal human CNS cells; (b) to characterize each clone with respect to cell-specific markers, receptors, major histocompatability complex (MHC) Class I and Class II antigens, karyotype and tumorigenicity; and (c) to study the mechanisms of differentiation and spontaneous and drug-induced degeneration, using a cholinergic clone as an experimental model. Our previous experience in establishing clonal lines of immortalized cells from fetal rat CNS tissue will be of great value for this study. The logistics of obtaining human fetal CNS tissues have already been estabiished. We propose to transfect freshly prepared single-cell suspensions of fetal CNS cells with plasmid vectors, pSV5-neo or psv5-neo, using an electroporation technique. The immortalized cells will be selected by crowing them in a medium containing a lethal concentration of qeneticin. The clones of various neurons and glia will be established by using a single-cell clonine method. A cholinergic clone will be selected to investiqate the mechanisms of differentiation and degeneration. Differentiated neurons degenerate spontaneously in vitro. Diverse agents such as prostaglandins and glutamic acid will be used to induce degeneration in neurons. Future studies will focus on elucidating the molecular mechanisms which regulate differentiation and degeneration in CNS neurons, and efficacy of these neurons in neural transplant studies.
