Gene targeting of the NKX2.1 locus facilitates the isolation of human forebrain progenitors from differentiating human embryonic stem cells

Key to the normal development of the vertebrate forebrain is the emergence of distinct progenitor zones that generate precursors to neural cell types that will eventually migrate to throughout the forebrain, establishing neural communication networks. Perhaps the most dynamic of these progenitor zones are those located at the ventral basal region of the telencephalon; a region that contains the ganglionic eminences. Within these, the medial ganglionic eminence (MGE) is the primary source of inhibitory GABAergic interneurons. Crucial to the development of the MGE is the expression of the homeobox transcription factor Nkx2.1, which functions initially in early ventral specification of the forebrain and is later involved in precursor migration as well as controlling GABAergic interneuron subtype differentiation. In order to generate a platform for investigating human forebrain development, we have used homologous recombination in hESCs to insert sequences encoding GFP into the NKX2.1 locus. GFP expression was demonstrated to faithfully report NKX2.1 expression, enabling the prospective isolation of human NKX2.1+ basal forebrain progenitors. Experiments with NKX2.1GFP/w hESCs demonstrated that the generation of NKX2.1-GFP+ cells was dependent on the concentration, timing and duration of retinoic acid treatment during differentiation. Gene expression studies and extended differentiation experiments performed with isolated NKX2.1-GFP+ cells indicated a multipotent nature of this precursor population.