Stem cells establish specialized cells, regenerate damaged tissues, and maintain homeostasis of short-lived cell populations, such as those of the skin and blood.1,2,3 The ability to produce both differentiated and undifferentiated cells is critical for tissue function and is dependent on signals from the local microenvironment. Neighboring cells and environmental niches produce signals that regulate whether a stem cell undergoes symmetric or asymmetric cell division (ACD). ACD produces one daughter cell that self-renews and one that undergoes differentiation. In contrast, symmetric cell division generates two identical daughter cells which either self-renew or differentiate.
Spatially oriented signals, such as those generated by environmental niches, stimulate ACD by polarizing cell fate determinants and regulating the position of the mitotic spindle.4 The orientation of the mitotic spindle directs the axis of cell division and controls the inheritance of cell fate determinants by the daughter cells.5,6 The lack of oriented signals in cell culture has limited the identification of signaling pathways that regulate ACD. However, this experimental limitation was recently overcome by Habib and colleagues who immobilized signaling molecules onto 2.8 μm beads through chemical conjugation. 7 The spatially restricted protein-conjugated beads deliver oriented signals to cultured cells, enabling assessment of ACD at the single cell level.
Wnt-3a, which stimulates the canonical Wnt signaling pathway, has been shown to affect cell polarity and fate in the nematode, Caenorhabditis elegans.8 In mammalian cells, a role for Wnt-3a in cell fate determination is suggested by its ability to maintain cultured mouse embryonic stem (ES) cells in an undifferentiated, pluripotent state.9,10,11 While uniform Wnt-3a signals maintain stem cell pluripotency in culture, the effect of oriented Wnt signals on cell fate and polarity has not been established. To investigate the role of oriented signals on stem cell division, Habib et al. introduced recombinant mouse Wnt-3a-conjugated beads to mouse ES cells and used live cell imaging to examine the effect of spatially-restricted Wnt-3a on the polarity of signaling molecules, alignment of the mitotic spindle, and expression of cell fate determinants.
Using live cell imaging of GFP-tagged proteins and immunostaining of untagged proteins, Habib and colleagues demonstrated that immobilized Wnt-3a induced asymmetric localization of Wnt signaling proteins including Frizzled-1, Adenomatous Polyposis Coli, Low-density Lipoprotein Receptor-related Protein-6, and beta-Catenin.7 Asymmetric localization led to a high concentration of Wnt signaling components proximal to immobilized Wnt-3a. In contrast, Wnt-5a-conjugated control beads did not induce significant asymmetric localization of Wnt signaling components.
Exposure of mouse ES cells to spatially restricted Wnt-3a resulted in alignment of the mitotic spindle in parallel with the axis of cell polarity that was generated by asymmetric localization of signaling molecules. The effect of Wnt-3a on mitotic spindle orientation and cell polarity resulted in ACD with one daughter cell inheriting significantly more Wnt signaling components than the other. Furthermore, the “Wnt-on” and “Wnt-off” phenotypes of the daughter cells led to different protein expression profiles and cell fates. Using GFP fused to the promoters of Rex-1/ZFP42, SOX2, and Stella/Dppa3, the “Wnt-on” cells were shown to express higher levels of transcripts corresponding to pluripotency markers compared to “Wnt-off” cells. The “Wnt-on” cells also demonstrated an increase in expression of protein markers of pluripotency. In contrast, Wnt-5a-conjugated control beads failed to induce asymmetric expression of pluripotentcy markers. Furthermore, the “Wnt-off” cells expressed higher levels of Claudin-6 and similar levels of Oct-4 compared to the “Wnt-on” cells, a phenotype that is consistent with epiblast stem cells. The more differentiated phenotype of the “Wnt-off” cells emphasizes the importance of Wnt-3a in maintaining stem cell pluripotency.
The findings by Habib et al. show that oriented Wnt-3a signals induce asymmetric division of mouse ES cells by concentrating Wnt signaling components to the side of the cell that is proximal to the Wnt signal. This polarization leads to a “Wnt-on” phenotype, which upon cell division, generates one pluripotent daughter cell and one daughter cell that is fated for differentiation. In contrast, when Wnt-3a signals are uniformly distributed within a cell, the entire cell adopts a “Wnt-on” phenotype and cell division results in two pluripotent daughter cells. These findings demonstrate a mechanism by which Wnt-3a maintains mouse ES cell pluripotency in culture and suggests that the presence of oriented Wnt-3a signals in vivo may induce asymmetric cell division.
Although the distribution of polarized Wnt signals in vivo is not known, it is tempting to speculate that the orientation of Wnt-3a signals generated by stem cell niches could regulate the balance between maintenance of stem cell populations and the production of differentiated cells. Manipulating pathways that regulate stem cell maintenance and differentiation in vitro could improve the maintenance, expansion, and differentiation of stem cell populations that are difficult to culture. Targeting similar pathways in vivo may present novel strategies to increase stem cell engraftment, encourage regeneration of damaged tissues, or restore activity to suboptimal stem cell populations.
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Spatially Restricted Wnt-3a Induces Asymmetric Division of Mouse Embryonic Stem Cells by Polarizing Cell Fate Determinants and Orienting the Mitotic Spindle. Exposure of embryonic stem cells to spatially restricted Wnt-3a signals induces polarized localization of canonical Wnt signaling proteins and alignment of the mitotic spindle in parallel with the axis of cell polarity. The polarity of Wnt signaling components combined with the orientation of the mitotic spindle results in asymmetric cell division. The daughter cell proximal to immobilized Wnt-3a demonstrates a “Wnt-on” phenotype and expresses markers of pluripotency such as Rex-1/ZFP42, SOX2, and Stella/Dppa3. The daughter cell distal to immobilized Wnt-3a demonstrates a “Wnt-off” phenotype and expresses markers of epiblast stem cells such as Claudin-6.
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