SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland

Abstract

The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (<0.05%) population of progenitor cells in the adult pituitary gland. We show that these cells express SOX2, a marker of several early embryonic progenitor and stem cell types, and form "pituispheres" in culture, which can grow, form secondary spheres, and differentiate to all of the pituitary endocrine cell types, as well as folliculostellate cells. Differentiation of cells in the pituispheres was associated with the expression of nestin, SOX9, and S100. Cells expressing SOX2 and E-cadherin are found throughout Rathke's pouch (RP) in embryos but persist in the adult gland, mostly in a narrow zone lining the pituitary cleft, but also are scattered throughout the pituitary. However, unlike in embryonic RP, most of these SOX2(+) cells in the adult gland also express SOX9 and S100. We suggest that this SOX2(+)/SOX9(+) population represents transit-amplifying cells, whereas the SOX2(+)/SOX9(-) cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.

Fig. 1.

SOX2 expression in embryonic and adult pituitary. (a) At 11.5 dpc, SOX2 is ubiquitous in RP, and cell membranes facing the pouch's lumen (L) show E-cad immunoreactivity. (b and c) At 16.5 dpc (b), a few ventral cells coexpress SOX2 and Pit1 (arrowheads), but SOX2 is excluded from the endocrine cell population in the anterior lobe at 18.5 dpc (c). (d) In the adult pituitary, no colocalization is found with Pit1, and SOX2⁺ cells in the cleft still express E-cad. (e) One and one-half hour BrdU labeling shows high mitotic activity in SOX2⁺ embryonic pituitary progenitors at 12.5 dpc. (f) In the adult, a few cells lining the pituitary cleft (PC) are still mitotically active. (g and h) SOX2 is not expressed in somatotrophs (g) but is expressed in marginal cells (between dashed lines) and FS cells as shown by its coexpression with S100 protein (h). AL, anterior lobe; IL, intermediate lobe; Horm, pituitary hormones. (Scale bars: 15 μm.)

Fig. 2.

SOX9 is expressed in the embryonic pituitary and defines two populations of SOX2⁺ cells in the adult gland. (a and b) At 12.5 dpc (a), SOX9 is only sparsely expressed within the RP but shows a strong colocalization with SOX2 at 18.5 dpc (b). At the earlier stage, there are mesenchymal cells (Mes) strongly positive for SOX9 outside the pouch. (c and d) In the adult cleft (c), SOX2 and SOX9 are coexpressed in marginal cells (between dotted lines) and FS cells, although a few SOX2⁺ cells, indicated by asterisks, are negative for SOX9 (d). L, Rathke's pouch lumen; A, anterior; P, posterior; PC, pituitary cleft. (Scale bars: 15 μm.)

Fig. 3.

The SOX2⁺ subpopulation forms pituispheres in culture. (a) BrdU labeling shows that pituispheres grow by cell division, not aggregation. (b and c) SOX2, E-cad (b), and Sca-1 (c) are highly expressed in 4-day-old pituispheres. (d–f) SOX2 and E-cad are still highly expressed after 7 days in culture (d), and an FS cell-like population appears as shown by S100β immunostaining (e), which coincides with the onset of SOX9 expression (f). (g) As SOX9 expression increases, the protein shows more nuclear localization. (h) When pituispheres are grown in differentiating conditions, SOX9 quickly becomes nuclear, and regions of higher SOX9 nuclear localization correlate with regions of S100β expression. (Scale bars: 5 μm.)

Fig. 4.

Pituispheres contain multipotent progenitors. (a) In the presence of growth factors, a few endocrine cells differentiate inside pituispheres, and some still express SOX2. (b) When 5-day-old pituispheres are cultured on Matrigel-coated coverslips in the absence of growth factors, endocrine cells differentiate within 4 days, although SOX2- and E-cad-expressing cells remain evident. (c) After 8 days, more hormone-producing cells are found mostly at the surface of the sphere, whereas S100β is found in regions within the structure. (d–h) GH (d), ACTH (e), LH (f), PRL (g), and TSH (h) immunostainings after 9 days in differentiating conditions. (i) Pituisphere cells can give rise to differentiated cells from separate lineages as shown by the presence of both GH- and ACTH-producing cells in the same pituisphere. (Scale bars: 5 μm.)