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A CNS-derived conditionally immortalized temperature-sensitive neural progenitor (CINP) cell line was used to generate NGF-secreting cells suitable for intracerebral transplantation. The cells were transduced by repeated retroviral infection, using a vector containing the mouse NGF cDNA under the control of the LTR promoter. Subcloning at the permissive temperature (33 degrees C) identified a highly NGF-secreting clone (NGF-CINP), which contained multiple copies of the transgene and released NGF at a rate of 2 ng/hr/10(5) cells in vitro, both at 33 and 37 degrees C, which was approximately 1 order of magnitude higher than what was possible to achieve in the heterogeneously infected cell cultures. After transplantation to the brain, the NGF-CINPs differentiated into cells with a predominant glia-like morphology and migrated for a distance of 1-1.5 mm from the implantation site into the surrounding host tissue, without any signs of overgrowth and tumor formation. Grafts of NGF-CINP cells implanted into the septum of adult rats with complete fimbria-fornix lesion blocked over 90% of the cholinergic cell loss in the medial septum and grafts placed in the intact striatum induced accumulation of low-affinity NGF receptor positive fibers around the implantation site. Expression of the NGF transgene in vivo was demonstrated by RT-PCR at 2 weeks after grafting. It is concluded that the immortalized neural progenitors have a number of advantageous properties that make them highly useful experimental tools for gene transfer to the adult CNS.

Previous studies suggest that a population of precursor cells from the developing and adult mouse striatum can be expanded in culture using serum-free, N2-supplemented medium and mitogenic factors such as epidermal growth factor (EGF). Here we show that EGF-responsive precursor cells from embryonic rat striatum and mesencephalon can also be expanded in culture, incorporate bromodeoxy uridine (BrDU) and develop into spheres that either adhere to the surface of the culture dish or float freely in the medium. Addition of B27, a medium supplement that increases neuronal survival in primary CNS cultures, resulted in a tenfold increase in the number of proliferating cells in vitro over the first week. The effects of B27-supplemented medium on precursor cell survival were only seen when primary cultures were used, such that dividing cells grown in B27 for 1 week could then be transferred to either B27 or N2 medium and show similar survival and division rates in response to EGF. After 1, 2 or 4 weeks of growth in B27-supplemented medium, dissociated precursor cells from either striatal or mesencephalic cultures could be differentiated when exposed to a poly-l-lysine-coated substrate in serum and EGF-free medium supplemented with B27. These cells then matured into a mixed culture containing neurons (approximately 35% of cells), astrocytes (approximately 44% of cells), and oligodendrocytes (approximately 10% of cells), based on immunocytochemical staining with microtuble-associated protein (MAP2), glial fibriallary acidic protein and galactocerebrosidase. When whole spheres of precursor cells were allowed to differentiate, every one examined was found to generate neurons, astrocytes and oligodendrocytes in similar proportions.(ABSTRACT TRUNCATED AT 250 WORDS)