In vitro expansion of neural progenitor cells followed by induction of dopaminergic phenotype may provide a limitless source of cells for grafting into patients with Parkinson's disease (PD). However, the signals controlling the conversion of these cells into dopamine (DA) neurons must be identified. In an effort to accomplish this, single cells isolated from ventral mesencephalon were clonally expanded and exposed to hematopoeitic cytokines and neurotrophic molecules. Analysis of cell differentiation in response to this treatment yielded conversion of a high percentage (72 to 98 percent) of cells in some clones to a tyrosine hydroxylase (TH)-positive phenotype. Of the 24 clones generated, the best conversion to TH cells occurred with exposure to a combination of interleukin-1 (IL-1), interleukin- 11 (IL-11), leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF). Positive clones expressed TH, the DA transporter, Nurr-1 and released DA in culture. Other cells in cytokine-exposed clones expressed GFAP (astrocyte marker) or MAP-2 (neuron marker) indicating that the original neurospheres were also capable of producing clones that differentiate into glial and nondopaminergic neurons. Initial neural grafting studies m the rat model of PD using a clone with the highest conversion rate to TH indicated that converted progenitor cell grafts produced complete amelioration of amphetamine-induced rotational behavior and continued to express the TH phenotype. However, the survival rate of these grafted progenitor cells was reduced (26 percent) compared to embryonic ventral mesencephalon (VM). The experiments proposed here will develop protocols for optimal survival of Wafted cytokine-converted mesencephalic progenitor cells. Once survival of grafted mesencephalic progenitor cells is optimized, direct comparisons will be made to fresh embryonic VM grafts on measures of behavior, in vivo dialysis, post-mortem DA biochemistry, DA receptors, cell survival and neurite extension. Lastly, this proposal will test the efficacy of the DA conversion cocktail on clonal progenitors derived from embryonic mesencephalon of nonhuman primate brain. If successful, cytokine-converted mesencephalic progenitor cells could potentially replace embryonic tissue as the primary source of cells for grafting in PD.
