A niche is defined as a microenvironment that maintains

A niche is defined as a microenvironment that maintains stem cisapride through growth factors, cell surface proteins and extracellular matrices (ECMs), and is identified in some organs such as the intestine, brain, skeletal muscle, skin and testis (Chen and Chuong, 2012; Gattazzo et al., 2014; Tanimura et al., 2011). In the adult anterior pituitary, the marginal cell layer (MCL) facing the residual lumen and SOX2-positive cell clusters scattered in the parenchyma of the anterior lobe are proposed as primary and secondary stem/progenitor cell niches, respectively, and the secondary niches originate from the primary niche (Gremeaux et al., 2012; Vankelecom and Chen, 2014). Interestingly, although the MCL-niche exists from early pituitary development to adult pituitary, the parenchymal-niche only appears after birth and increases in number during the early postnatal pituitary growth wave (Chen et al., 2013). In addition, our previous studies demonstrated that stem/progenitor cells construct both niches by a homophilic tight junction factor, CAR (coxsackievirus and adenovirus receptor encoded by the Cxadr gene) (Chen et al., 2013) and a juxtacrine factor, ephrin-B2 (Yoshida et al., 2015). We also showed that PROP1/SOX2-positive cells are significantly decreased in the MCL during the early postnatal pituitary growth wave, but are still maintained in the parenchymal-niche (Higuchi et al., 2014; Yoshida et al., 2011). Considering that S100β-positive stem/progenitor cells appear only after birth and the quantitative and qualitative transition of stem/progenitor cells occurs during early postnatal periods, the parenchymal-niche might play a role in the cell turnover required for the postnatal anterior pituitary. A sphere-forming assay is a valuable method for analyzing the characteristics of stem/progenitor cells after forming a sphere originating from a single stem/progenitor cell in various tissues as well as in the pituitary (Chen et al., 2005; Fauquier et al., 2008; Gritti et al., 1996; Pastrana et al., 2011; Reynolds and Weiss, 1992). In addition, analysis of the side population, which is a fraction rich in stem/progenitor cells, have demonstrated several candidate regulators of stem/progenitor cells such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (Chen et al., 2006; Chen et al., 2005; Fauquier et al., 2008). However, in the pituisphere and side population, it is difficult to distinguish whether SOX2-positive stem/progenitor cells are derived from the MCL-niche, the parenchymal-niche, or a single-SOX2 positive cell detached from the niches in the parenchyma. Therefore, isolation and characterization of the pituitary stem/progenitor cells of known origin provide useful information to clarify the mechanisms for regulating stem/progenitor cells in their niche.
Materials and methods
Results
Discussion Recent reports proposed that SOX2-positive stem/progenitor cells of the adult anterior lobe form two types of niche, the MCL and the scattered dense cell clusters in the parenchyma (Chen et al., 2013; Gremeaux et al., 2012; Yoshida et al., 2015). Especially, the appearance of the parenchymal-niche only after birth (Chen et al., 2013) indicates that the parenchymal-niche plays key roles in the cell turnover required for the postnatal anterior pituitary. However, this hypothesis has not been verified, because we lacked appropriate methods for isolating the pituitary stem/progenitor cells from the parenchymal-niche. In this study, we aimed to isolate stem/progenitor cells from the parenchymal-niche and succeeded in isolating the dense cell clusters originating from the parenchymal-niche, termed PS-clusters, by taking advantage of its tight structure resistant to protease treatment. The PS-clusters were composed of three subtypes based on S100β-GFP. Their stemness was confirmed by the presence of several stem/progenitor cell markers and their differentiation capacity by in vitro differentiation assay.