A side population (SP) has been identified in a number of tissues, where it typically represents a small population enriched in stem/progenitor cells. In this study we show that the adult mouse anterior pituitary (AP) also contains a characteristic SP displaying verapamil-sensitive Hoechst dye efflux capacity. A majority of the SP cells express stem cell antigen 1 at a high level (Sca1high). Using (semi)quantitative RT-PCR and immunofluorescence, we characterized the Sca1high SP as a population enriched in cells expressing stem/progenitor cell-associated factors and components of the Notch, Wnt, and sonic hedgehog signaling pathways, functional in stem cell homeostasis as well as in early pituitary embryogenesis. Lhx4, a transcription factor pivotal for early embryonic development of the AP, was only detected in the Sca1high SP, whereas Lhx3, in contrast to Lhx4 not down-regulated after AP development, was only found in the main population. The Sca1high SP was depleted from cells expressing phenotypic markers of differentiated AP cells (hormones), but contained a small proportion of folliculo-stellate cells. Stem cells of many tissues can clonally expand to nonadherent spheres in culture. Clonal spheres also developed in AP cell cultures. Spheres showed an expression pattern resembling that of Sca1high SP cells. Moreover, the sphere-initiating cells of the pituitary segregated to the SP and not to the main population. In conclusion, we show that the adult pituitary contains a hitherto undescribed population of cells with SP phenotype and clonal expansion capacity. These cells express (signaling) molecules generally found in stem/progenitor cells and/or operative during pituitary early embryonic development. These characteristics are supportive of a stem/progenitor cell phenotype.

The pituitary gland represents the endocrine core, governing the body's hormonal landscape by adapting its cellular composition to changing demands. It is assumed that stem/ progenitor cells are involved in this remodeling. Recently, we uncovered a candidate stem/progenitor cell population in the anterior pituitary. Here, we scrutinized this ''side population'' (SP) and show that, unexpectedly, not the subset expressing high levels of ''stem cell antigen-1'' (Sca1 high ) but the remainder non-Sca1 high fraction clusters the pituitary progenitor cells. Transcriptomal interrogation revealed in the non-Sca1 high SP upregulated expression of the pituitary stem/progenitor cell markers Sox2 and Sox9, and of multiple factors critically involved in pituitary embryogenesis. The non-Sca1 high SP encloses the cells that generate spheres and display multipotent hormone differentiation capacity. In culture conditions selecting for the nonSca1 high subset within the SP, stem cell growth factors that induce SP expansion, affect transcription of embryonic factors, suggesting impact on a developmental program that unfolds within this SP compartment. Non-Sca1 high SP cells, revealed by Sox2 expression, are observed in the postulated periluminal stem/progenitor cell niche, but also in small groups scattered over the gland, thereby advocating the existence of multiple niches. In early postnatal mice undergoing a pituitary growth wave, Sox2 1 cells are more abundant than in adults, concordant with a larger SP and higher non-Sca1 high proportion. Together, we tracked down pituitary progenitor cells by SP phenotype, and thus provide a straightforward method to isolate and scrutinize these cells from the plastic pituitary ex vivo, as well as a culture system for in-depth exploration of their regulatory network.

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Nestin is an intermediate filament protein that has originally been identified as a marker of neuroepithelial stem/progenitor cells. The present study explored whether nestin immunoreactivity (nestin-ir) is present in the rat pituitary and in which cell type(s). Nestin-ir was observed in scattered cells in the anterior, intermediate, and neural lobes. Nestin-ir cells were predominantly of stellate shape and were more numerous in immature than in adult animals. Nestin-ir did not colocalize with any pituitary hormone, and did not colocalize or only very sporadically with the folliculo-stellate cell marker S100. In the intermediate lobe, nestin-ir cells contained glial fibrillary acidic protein in an age-dependent manner. Nestin-ir cells were closely associated with endothelial and fibronectin-ir cells, but did mostly not coincide. Nestin-ir was not found in alpha-smooth muscle actin-ir myofibroblasts or in microglial cells. Regardless of age, nestin-ir was detected in some unidentifiable cells that border the pituitary cleft. Nestin-ir remained present in pituitary cultured as three-dimensional aggregates. Treatment with basic fibroblast growth factor or leukemia inhibitory factor increased the number of nestin-ir cells. Starting from anterior lobe cell monolayer cultures, nestin-ir cells could be selected and propagated to a virtually pure population. These nestin-ir cells displayed remarkable motility and proliferative activity, and did not express hormones, glial fibrillary acidic protein, or S100, but contained vimentin-, fibronectin-, and alpha-smooth muscle actin-ir. In conclusion, nestin-ir is present in the pituitary in cells that are neither hormonal nor typical folliculo-stellate. The expression pattern depends on age and lobe examined. Pericapillar localization suggests a pericyte phenotype for some of them. Whether the heterogeneous nestin-ir population also contains pituitary progenitor cells remains to be explored.