Recent studies have demonstrated that Sox2-expressing stem/progenitor cells play roles in the pituitary cell turnover. Two types of niches have been proposed for stem/progenitor cells, the marginal cell layer (MCL) and the dense cell clusters in the parenchyma. Among them, the appearance of the parenchymal-niche only after birth indicates that this niche is involved in the cell turnover required for the postnatal pituitary. However, little is known about the roles of the parenchymal-niche and its regulation. The present study aimed to isolate pituitary stem/progenitor cells from the parenchymal-niche in the adult rat pituitary. Cell dispersion by stepwise treatment with proteases allowed the isolation of dense cell clusters. Immunocytochemistry demonstrated that clusters are universally composed of SOX2-positive cells, and most of them are positive for PROP1. Taken together with the anatomical analysis, we concluded that the isolated clusters are the parenchymal stem/progenitor cell (PS)-clusters, not the MCL-one. PS-clusters cultivated by serum-free overlay 3-dimensional culture maintained their stemness, and treatment with bFGF and EGF induced cyst-formation. Moreover, PS-clusters demonstrated some differentiation capacity with GSK3β-inhibitor treatment. Collectively, the present study demonstrates a simple method for isolating stem/progenitor cells from the parenchymal-niche, and provides tools to analyze the factors for regulating the pituitary niches.

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Paired-related homeobox transcription factors, PRRX1 and PRRX2, are known to be important factors for craniofacial and limb morphogenesis. We recently cloned Prrx2 from the porcine adult pituitary cDNA library and found that only PRRX1 is present in the rat embryonic pituitary. In this study, we re-investigated the temporospatial expression and localization of PRRX1 and PRRX2 in the rat pituitary throughout life. The persistent expression of Prrx1 was ascertained after the middle stage of embryonic development, whereas significant expression of Prrx2 was found only in the postnatal pituitary. Immunohistochemistry confirmed that PRRX1-positive cells appeared inside the pituitary on embryonic day 16.5 in the marginal cell layer (MCL), a pituitary stem/progenitor cell niche, and the expanding parenchyma of the anterior pituitary. In contrast, PRRX2-positive cells first appeared in the anterior lobe and intermediate lobe sides of the MCL around postnatal day 30 when the postnatal pituitary growth wave had almost terminated. Immunostaining for PRRX1 with a stem/progenitor cell marker SOX2, a pituitary progenitor marker PROP1, or pituitary hormones revealed that PRRX1 localized in cells in the transition process from the multipotent progenitor stage to the early stage of terminal differentiation throughout life. PRRX2 emerged in cells positive for SOX2 but negative for PROP1 in the anterior and intermediate lobe sides of the postnatal MCL. Thus, PRRX1 and PRRX2 might participate distinctly in pituitary organogenesis and the postnatal cell-supply system.

We have recently shown that cells positive for the paired-related homeobox transcription factors PRRX1 and PRRX2 occur in the rat pituitary, and that they are derived from two different origins: pituitary-derived cells positive for stem cell marker SOX2 and extra-pituitary-derived cells negative for SOX2. In this study, we have further characterized the PRRX1- and PRRX2-positive cells that originate from extra-pituitary cells. Immunohistochemical analyses were performed with specific antibodies against PRRX1 and PRRX2 in order to clarify their roles in pituitary vasculogenesis. PRRX1- and PRRX2-positive cells were found in Atwell's recess and at the periphery of the pituitary on embryonic day 15.5 (E15.5). Several PRRX1-positive cells then invaded the anterior lobe, together with a few PRRX2-positive cells, on E16.5. Some PRRX1-positive cells were also positive for mesenchymal stem cell marker NESTIN. Moreover, some PRRX1/NESTIN double-positive cells showed characteristics of vascular endothelial cells with an Isolectin-B4-binding capacity. PRRX1 co-localized with vascular smooth muscle cell/pericyte marker α-smooth muscle actin in the deep area of Atwell's recess. We confirmed the presence of PRRX2/NESTIN double-positive cells at an entry area in Atwell's recess and at the periphery of the pituitary, but PRRX2 did not co-localize with Isolectin B4 or α-smooth muscle actin. These data suggest that PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are present at the periphery of the embryonic pituitary and at the entry from Atwell's recess and participate in pituitary vasculogenesis by differentiation into vascular endothelial cells and pericytes, whereas the presence of PRRX2 indicates much higher stemness than PRRX1.