The in vitro neuralization of hESCs has been widely used to generate central and peripheral nervous system components from neural precursors (Bajpai et al., 2009; Curchoe et al., 2010), most often through an intermediate "rosette" stage. Here we confirm that hESC derived neuro-epithelial rosettes express many characteristics of the developing embryonic neural plate (Aaku-Saraste et al., 1996), characterized by expression of the tight junction proteins ZO-1 and N-Cadherin. Moreover, neuro-epithelial rosettes display a characteristic acetylated alpha tubulin cytoskeletal arrangement (similar to that observed in the developing embryonic neural plate) (Bhattacharyya et al., 1994). Demonstrated here for the first time MKLP was observed in a hESC model system. We found MKLP expression in small particles in between mitotic spindles, large particles aggregating in the lumen of neuroepithelial rosettes, and we did not observe MKLP in the nucleus of hESC derived neural precursors as previously described in the HeLa cell line. We observed MKLP+ particles in aggregations in the lumen of "early" rosette structures. Furthermore, we observed that MKLP+ particle aggregations can also be lost from the lumens of hESC derived neuro-epithelial rosettes, similar to a phenomenon observed in the developing neural tube in vivo (Marzesco et al., 2005). We determined that this loss of MKLP+ particles occurs from "late" as opposed to "early" stage neuro-epithelial rosettes (characterized by junction type). Disrupting the apical-basal polarization of "early" stage rosettes with a 1% Matrigel overlay (Krtolica et al., 2007) nearly ablates MKLP particle aggregation in the lumen of rosettes, demonstrating that the apical-basal polarity of early NE cells is necessary for lumenal MKLP particle aggregation. We conclude that early hESC derived neuro-epithelial rosettes can model early neurulation events, such as the transition from neural plate like cells to neural tube like cells (i.e., symmetric to asymmetric NE cell division) demonstrated by polarized MKLP particle inheritance and distribution using junction type as a measure of stage.
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