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    • In the parenchymal niche found in the adult anterior

    2018-11-02

    In the parenchymal-niche found in the adult anterior lobe, SOX2-positive b-raf inhibitors were more densely connected to each other than in the MCL-niche, and formed dense cell clusters by E-cadherin (Fig. 1). We previously demonstrated that CAR and ephrin-B2 specifically localize at the apical cell membrane and intercellular surface of the dense cell clusters in the parenchyma, while in the MCL, they mainly localize at apical cell membranes facing the lumen (Chen et al., 2013; Yoshida et al., 2015). Further analyses demonstrated that, in the adult pituitary, PROP1/SOX2-double positive cells compose approximately 5.8% of SOX2-positive cells in the MCL, whereas about 90% of the parenchymal-niches of the anterior lobe contain PROP1-positive cells (Yoshida et al., 2015; Yoshida et al., 2011). Immunocytochemical images of E-cadherin, CAR and ephrine-B2 in the PS-clusters corresponded well to histological images observed in the parenchyma of the anterior lobe. Notably, we clearly showed that about 92% of PS-clusters contain PROP1-positive stem/progenitor cells. Taken together with a similarity of morphology between the isolated-dense cell clusters and parenchymal-niche, we concluded that dense cell clusters isolated in this study are originated from the parenchymal-niche, not the MCL-niche in the anterior lobe. Although both the MCL- and parenchymal-niches are similarly composed of SOX2- and E-cadherin-positive cells, the present method using trypsin and collagenase isolated only the stem/progenitor cells from the parenchymal-niche, not the MCL-niche. It is known that some stem cell niches are composed of unique ECMs. Heparan sulfate proteoglycans (HSPGs) are required for restriction of gremlin stem cells in the niches in Drosophila (Hayashi et al., 2009). In the neural stem cell niches (e.g. the subventricular zone and subgranular zone), heparan sulfate- and chondroitin sulfate-proteoglycans regulate the proliferation and differentiation of neural stem cells in the presence of growth factors, such as epidermal growth factor and fibroblast growth factor (Akita et al., 2008). In the hair follicle stem cell niche, a hemidesmosomal transmembrane collagen, collagen XVII, plays important roles in the maintenance of both hair follicle stem cells and melanocyte stem cells (Tanimura et al., 2011). Considering these findings, our present data suggest that SOX2-positive cell clusters scattered in the parenchyma of the anterior lobe might be constructed by the production of unique ECMs that differ from the MCL. In the present study, PS-clusters showed a lower differentiation capacity than pituispheres. In general, a sphere is formed by activating a single proliferative stem/progenitor cell using bFGF, EGF, B27- and N2-serum free supplements, which are suitable for cultivation of neural stem cells (Gritti et al., 1996; Pastrana et al., 2011; Reynolds and Weiss, 1992). Spheres are also known to lack quiescent stem cells (Pastrana et al., 2011). Indeed, although about 10% of the cells in the anterior lobe of an adult mouse are positive for SOX2, the proportion of cells showing pituisphere forming capacity is only about 0.2% of the dispersed cells from the adult mouse anterior lobe (Gremeaux et al., 2012). This clearly shows that only a small number of SOX2-positive cells are actively undergoing proliferation and differentiation. However, in the present study, we performed a differentiation assay using randomly selected PS-clusters. Considering that most of the SOX2-positive cells are in a quiescent state in the adult pituitary (Andoniadou et al., 2013), one of the reasons for these differences in the differentiation capacity between PS-clusters and pituispheres is the presence of quiescent SOX2-positive cells. In addition, SOX2-positive cells have sub-populations according to the existence of transcription factors and S100β (Higuchi et al., 2014; Yoshida et al., 2011). In the present study, using the S100β/GFP-TG rat model, we demonstrated that three subtypes of PS-clusters based on S100β-GFP signals can be isolated, and that null-GFP-clusters show different properties compared to GFP- and mixed-GFP-clusters under the culture conditions utilized (Fig. 5). Accumulating reports have demonstrated that S100β-positive cells produce several paracrine factors such as bFGF and leukemia-inhibitory factor, which are survival and/or growth factors for pituitary stem/progenitor cells (Chen et al., 2006; Denef, 2008). They also produce transforming growth factor β1, interleukin-6 and vascular endothelial growth factor of which effects for pituitary stem/progenitor cells have not yet been identified (Denef, 2008). To our knowledge, this study is the first to demonstrate the different property of SOX2-positive cells based on the existence of S100β. Our data suggest that SOX2-positive cells are heterogeneous, and a subset of these cells plays different roles such as supporting other types of SOX2-positive cells. Further studies involving comparative analysis of GFP- and null-GFP-clusters might elucidate their hierarchy in the process of differentiation, as well as the presence of niche cells.