Isolation of PRRX1-positive adult pituitary stem/progenitor cells from the marginal cell layer of the mouse anterior lobe.

We recently suggested that paired-related homeobox 1 (PRRX1) and sex-determining region Y-box 2 (SOX2) double-positive (PRRX1/SOX2-positive) cells are pituitary stem/progenitor cells. The present study aimed to isolate PRRX1-positive cells located in the pituitary stem/progenitor cell niche. Immunohistochemical analysis revealed that PRRX1/SOX2-positive cells were located mainly in the marginal cell layer (MCL)-niche of the adult mouse pituitary gland, but were rarely present in the parenchymal-niche. Two-dimensional cultivation of primary cells from the anterior lobe (AL), including the MCL-niche, achieved efficient proliferation of PRRX1/SOX2-positive cells with high expression levels of pituitary stem/progenitor cell and niche markers. In contrast, primary cells from the AL, excluding the MCL-niche, showed limited growth. When isolated PRRX1/SOX2-positive cells and clusters of the parenchymal-niche were compared immunocytochemically, aquaporin 5, a marker of the MCL-niche, was detected only in isolated cells. Three-dimensional cultivation of isolated PRRX1/SOX2-positive cells promoted the formation of cystic-like spheroids and differentiation into endocrine cells. Thus, PRRX1-positive adult pituitary stem/progenitor cells were successfully isolated from the MCL-niche. The present study provides a powerful tool to analyze the cell supply system in the pituitary gland.