Demonstrated here for the first time MKLP was observed in

Demonstrated here for the first time, MKLP was observed in a hESC model system. Small MKLP particles are observed between mitotic spindles, large MKLP+ particles are observed aggregating in the lumen of neuroepithelial rosettes, and MKLP was not observed in the nucleus of hESC derived neural precursors using this particular antibody, as previously described in (human) HeLa plk inhibitor (Liu and Erikson, 2007) and insect cells (Deavours and Walker, 1999) using GFP-fusion transfection. We characterize the epithelial junctions of the early, symmetrically dividing stage through the later, neuron generating stages of differentiation and demonstrate the importance of polarity and junction type in the phenomenon. This system can be used to study early neurulation events, such as the mechanism of MKLP particle loss form the lumen (not directly observed here) and apical membrane reduction in vitro, providing insight into one of the important cellular mechanisms of stem and progenitor cell maintenance.
Materials and Methods
Results
Discussion The in vitro neuralization of hESCs has been widely used to generate central and peripheral nervous system components from neural precursors, most often through an intermediate “rosette” stage (Schulz et al., 2003; Elkabetz et al., 2008). The earliest stages of human development are the least accessible and therefore, hESC culture represents a unique opportunity to study little known developmental phenomenon (Fasano et al., 2010). In this study we confirm that hESC derived neuro-epithelial rosettes (differentiated with our rapid differentiation protocol (Bajpai et al., 2009; Curchoe et al., 2010; Cimadamore et al., 2009)), express some characteristics of the developing embryonic neural plate, characterized by expression of the tight junction proteins ZO-1 and N-Cadherin (Aaku-Saraste et al., 1996). Moreover, neuroepithelial 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 by immunofluorescence. In our hands small MKLP particles are observed between mitotic spindles, large particles aggregating in the lumen of neuroepithelial rosettes, and was not observed in the nucleus of hESC derived neural precursors as previously described in (human) HeLa cells (Liu and Erikson, 2007) and insect cells (Deavours and Walker, 1999). This observation could be due to the differences between the two model systems or the method of detection used (GFP fusion protein transfections versus immune-fluorescence) and should be examined methodically. MKLP+ particles were observed in aggregations in the lumen of “early” rosette structures. Furthermore, we observed numerous free floating MKLP+ particles and empty lumen structures of hESC derived neuro-epithelial rosettes that had been maintained in culture for extended time periods and have lost either ZO-1 or N-Cadherin staining, similar to the phenomenon observed in the developing neural tube in vivo. We determined that the loss of MKLP+ particles occurs from “late” as opposed to “early” stage neuro-epithelial rosettes (characterized by junction type). Unfortunately, it was beyond the scope of this work to investigate the mechanism of MKLP+ particle release; however we have included some tantalizing videos in the supplemental data of MKLP+ particles in, what we can only assume, is the process of release into the media. In Supplemental Video 1, taken between days 3–5 of adherent cell culture, we observed MKLP+ particles to be moving slightly, apparently still associated with the neuroepithelial cells or culture dish. In Supplemental Video 2, taken after 5 days of culture, we observed MKLP+ particles floating freely. 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. In our hands the Matrigel overlay did not fully disrupt polarity and in some cases we observed intact rosettes. This could be due to the inherent heterogeneity of neurosphere cultures, as they tend to differ in size, and therefore volume at the time of adherence. We observed differing patterns of N-Cadherin (Supplemental Video 3) and ZO-1 (Supplemental Video 4) expression, of the membranes formed on the culture dish side and both sides of the lumen respectively. It is possible that the 1% Matrigel overlay disrupts molecular signaling from the disk of ZO-1 at the top of the neuroepithelial lumen.