To optimize neuronal graft survival in the CNS, fetal donor tissue is essential. Given the constraints of obtaining human fetal tissue, we undertook an alternative strategy to replace damaged CNS neurons. We developed 2 temperature sensitive cells lines (RN333, RN46A). At non- permissive temperature (38.5 degrees C), these cells differentiate with neuronal properties and their survival and differentiation in vitro can be regulated by target-derived signals. Following transplantation into adult CNS, similar target-regulated differentiation was observed. Moreover, recent results have identified neurotransmitters in both cell lines. Given these results, the following hypotheses were developed: 1. Because of multiple variables which interact to influence neuronal maturation, differentiation of a neuronal cell line will be maximal in vivo, where substrate cues, neuronal trophic factors, and tissue topography present an optimal environment for that differentiation. 2. The adult CNS retains the capacity to direct specific differentiation of neuronal precursors in a morphological direction consistent with that of endogenous neurons, provided that appropriate neuronal cell lines with homologous growth requirements are used for transplantation. 3. We contend that CNS-derived neuronal cell lines, such as the ones detailed in the present proposal, can replace damaged endogenous neurons following transplantation into adult CNS. The present proposal will test these hypotheses by addressing 3 specific issues. l) What are the growth factors which regulate RN33B cell survival and differentiation? 2) What are the """"""""rules"""""""" which govern the survival and differentiation of transplanted RN33B cells? These 2 specific aims will give insight into the trophic requirements for survival, plasticity, and differentiation of RN33B cells in vivo, as well as the ability of the adult CNS to direct that differentiation. 3) We will characterize in vitro and in vivo the RN46A cell line, which similarly differentiates neuronally and has constitutive serotonergic properties. Our ultimate goal is to understand the factors involved in the survival and differentiation of transplanted neuronal cell lines so that we can predict how a given cell line will behave when transplanted to a defined region of the CNS under specific conditions.
