Promoter-based isolation and fluorescence-activated sorting of mitotic neuronal progenitor cells from the adult mammalian ependymal/subependymal zone

Abstract

Neuronal precursor cells are widespread in the subependyma of the forebrain ventricular lining, and may provide a cellular substrate for brain repair. We have previously identified and isolated them from fetal brain, by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein (hGFP), driven by the early neuronal promoter for Talpha1 tubulin (P/Talpha1). Fetal neuronal precursors were thereby identified and harvested with both a high degree of enrichment, and a virtual abolition of glial contaminants. We have now extended this approach to include the isolation and purification of neuronal progenitors from the adult brain. Dissociates of the lateral ventricular wall, that included the combined ependymal/subependymal zone, were obtained from 3-month-old adult rats. These cells were cultured and transfected with P/Talpha1:hGFP plasmid DNA. Two days later, the cells were redissociated, sorted on the basis of Talpha1-driven GFP expression, and replated. The majority of these cells expressed the early neuronal proteins Hu and TuJ1/betaIII-tubulin upon FACS; within the week thereafter, most matured as morphologically-evident neurons, that coexpressed betaIII-tubulin and MAP-2. Fewer than 5% expressed astrocytic markers, compared to over half of the cells in matched samples that were either not sorted, or sorted after transfection with a plasmid bearing the nonfluorescent lacZ gene under the control of P/Talpha1 tubulin. Thus, the use of a fluorescent transgene under the control of an early neuron-selective promoter permits the enrichment of neuronal progenitor cells from the adult rat brain, in a form that may allow their heterologous implantation.