Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters

Many tissues arise from pluripotent stem cells through cell-type specificat ion and maturation. In the bone marrow, primitive stem cells generate all t he different types of blood cells via the sequential differentiation of inc reasingly committed progenitor cells. Cell-surface markers that clearly dis tinguish stem cells, restricted progenitors, and differentiated progeny hav e enabled researchers to isolate these cells and to study the regulatory me chanisms of hematopoiesis. Neuronal differentiation appears to involve simi lar mechanisms. However, neural progenitor cells that are restricted to a n euronal fate have not been characterized in vivo, because specific cell-sur face markers are not available. We have developed an alternative strategy t o identify and isolate neuronal progenitor cells based on dual-color fluore scent proteins. To identify and isolate directly progenitor cells from brai n tissue without the need for either transfection or intervening cell cultu re, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanc ed yellow fluorescent protein (EYFP) in neuronal progenitor cells and neuro ns under the control of the T alpha1 alpha -tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cell s under the control of the enhancer/promoter elements of the nestin gene. B y crossing these lines we obtained mice expressing both transgenes. To isol ate neuronal progenitor cells directly from the developing brain, we used f low cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study t he mechanisms of neurogenesis and to develop cell-based therapies for neuro logical disorders. J. Neurosci. Res. 65:220-227, 2001. (C) 2001 Wiley-Liss, Inc.