Genetic approaches identify adult pituitary stem cells

Abstract

Adult tissues undergo continuous cell turnover in response to stress, damage, or physiological demand. New differentiated cells are generated from dedicated or facultative stem cells or from self-renewing differentiated cells. Here we describe a different stem cell strategy for tissue maintenance, distinct from that observed for dedicated or facultative stem cells. We report the presence of nestin-expressing adult stem cells in the perilumenal region of the mature anterior pituitary and, using genetic inducible fate mapping, demonstrate that they serve to generate subsets of all six terminally differentiated endocrine cell types of the pituitary gland. These stem cells, while not playing a significant role in organogenesis, undergo postnatal expansion and start producing differentiated progeny, which colonize the organ that initially entirely consisted of differentiated cells derived from embryonic precursors. This generates a mosaic organ with two phenotypically similar subsets of endocrine cells that have different origins and different life histories. These parallel but distinct lineages of differentiated cells in the gland may help the maturing organism adapt to changes in the metabolic regulatory landscape.

Fig. 1.

Nestin-GFP cells in the adult and developing pituitary. (A) General view of adult mouse pituitary. PL, posterior lobe of pituitary; IL, intermediate lobe; AL, anterior lobe. An arrow indicates the lumen that separates intermediate and anterior lobes. The perilumenal area is a remnant of embryonic primordium of the anterior pituitary, Rathke's pouch. (B) In the pituitary of nestin-GFP transgenic mice the majority of GFP-positive cells are located in the perilumenal area (arrows). Several GFP-positive cells are seen at the border separating intermediate and posterior lobes (arrowhead). (C–F) GFP-positive cells express Lhx3 (C, arrows), EpCAM (D), and Sox2 and cytokeratin 8 (E). GFP expression was also found in the cells with long processes that were dispersed throughout the gland and did not express Lhx3 (F, arrow). [Scale bars: 400 μm (A), 35 μm (B) and 10 μm (C–F).] (G) Nestin-GFP-positive cells in the primordium of the anterior pituitary, Rathke's pouch (RP; Di, ventral diencephalon), are first detected at embryonic day 11.5 (Left). They localized in the upper portion of Rathke's pouch and express epithelial markers, such as EpCAM (Right, arrows). [Scale bars: 50 μm (Left) and 12.5 μm (Right).] (H) At p0, GFP-positive cells are seen almost exclusively in the perilumenal area of pituitary (Upper Left) and are Lhx3-positive (Upper Right), cytokeratin 8-positive (Lower Left), and EpCAM-positive (Lower Right). [Scale bars: 75 μm (Upper Left) and 15 μm (Upper Right and Lower).] (I) A significant number of GFP-positive cells is seen in pituitary parenchyma at p7 (Upper Left) at the beginning of the postnatal wave of the pituitary growth. These cells express cytokeratin 8 (Upper Right, arrows), Lhx3 (Lower Left, arrows), and EpCAM (Lower Right, arrows). (J) Proliferation of transgene-positive cells. At p0, transgene-positive cells located in perilumenal zone are negative for Ki67, a marker of proliferation (Left, arrowheads). Later, during a postnatal wave of pituitary growth nestin-GFP-positive cells that migrate into glandular zone are often positive for Ki67, a marker of proliferation (Center, arrows, p7). At p21, the majority of transgene-positive cells reside in the perilumenal zone. They are negative for Ki67 (Right, arrowhead). (Scale bars: 15 μm.)

Fig. 2.

Nestin-expressing precursors/stem cells contribute to all six terminally differentiated cell types in the anterior pituitary. (A) GFP-positive cells are seen in the brain and pituitary gland of newborn (p0) nestin-cre recombinase transgenic mice crossed with the ROSA-lsl-GFP reporter strain. H, hypothalamus; AP, anterior pituitary; RT, rostral tip of the anterior pituitary. In the anterior pituitary, a few cells express GFP. The majority of rostral tip thyrotrophs are GFP-positive. Note that the majority of the differentiated neurons in the hypothalamus are GFP-positive, which indicates the effectiveness of Cre-induced recombination in neural stem cells. The expression of GFP in this particular mouse reporter line is weak but can be effectively visualized with anti-GFP antibodies. (Scale bar: 75 μm.) (B) Accumulation of GFP-positive cells in the anterior pituitary of nestin-Cre transgenic mice crossed with ROSA-lsl-GFP reporter mice. The number of GFP-positive cells reaches a peak around age 5 months. Female pituitaries (Upper) contain significantly higher numbers of GFP-positive cells than males (Lower). (Scale bar: 400 μm.) (C) Accumulation of nestin-Cre-derived cells in the anterior pituitary is age- and sex-dependent. The percentage of GFP-expressing cells gradually increases between 2.5 and 5 months. Between ages 5 and 8 months, a significantly higher number of stem cell-derived differentiated cells was found in the female anterior pituitary. Male pituitaries contain almost twice the percentage of somatotrophs in comparison with lactotrophs, whereas female pituitaries contain many more lactotrophs than somatotrophs. Correspondingly, the majority of GFP-positive progeny of nestin-expressing stem cells in males differentiated into somatotrophs, whereas in female most of them became lactotrophs. (D) GFP-positive cells contribute to each endocrine lineage: melanotrophs in the intermediate lobe and corticotrophs in the anterior lobe, visualized with anti-MSH and anti-ACTH antibodies correspondingly; gonadotrophs (anti-αGSU antibody); and Pit-1-dependent cell lineages (anti-Pit-1 antibody) that include somatotrophs (anti-GH antibody), lactotrophs (anti-PRL antibody), and thyrotrophs (anti-TSHβ antibody). Yellow shows colocalization of transgene and corresponding marker. (Scale bar: 30 μm.) (E) Nine-month-old mice carrying Cre-ER fusion protein driven by the nestin regulatory elements and the ROSA-lsl-GFP transgene were treated with tamoxifen twice with a 24-h interval and killed 1 month later. Expression of GFP reporter was found in perilumenal precursors as well as in terminally differentiated cells—ACTH-positive, αGSU-positive, and Pit-1-positive cells (fourth image). [Scale bars: 60 μm (first image) and 30 μm (other images).]

Fig. 3.

Nestin-GFP-positive cells can self-renew and differentiate along all pituitary lineages in vitro. (A) Epithelial colonies grown from nestin-GFP transgenic mice undergo spontaneous differentiation after exiting from exponential growth. At day 8, the majority of the cells still express EpCAM (A) and Sox2 (B) as Sox2 and Lhx3/nestin double positive cells (A–C). Cell aggregates often contain ACTH-positive cells (D), dispersed αGSU-positive cells (E), and rare Pit-1-positive cells (F) increasing at day 12. (G) GH-positive somatotrophs. (H) PRL-positive lactotrophs. (I) TSHβ-positive thyrotrophs. (Scale bars: A, 70 μm; B, 35 μm; C and F, 100 μm; D and E, 10 μm; the bar in E corresponds to 5 μm in G–I.

Fig. 4.

Nestin-GFP-positive cells in the pituitary tumor. (A and B) Pituitary tumor in a 12-month-old mouse that was Rb+/− and carried the nestin-GFP transgene. A macro view (A) and section of the organ (B, H&E) show that the tumor consisted of multiple foci of endocrine proliferating cells arisen in the intermediate lobe that compressed both posterior and intermediate lobes. Divided cells are frequently seen in these nodules (B, H&E). Tu, tumor. [Scale bars: 400 μm (Left) and 20 μm (Right).] (C–F) Distribution and phenotype of nestin-GFP-positive cells inside pituitary tumor. Nestin-GFP-positive cells are located mostly on the periphery of tumor nodules (C). They are positive for Lhx3, EpCAM (D), Sox2 (E), and keratin 8 (F), but, in contrast to the majority of tumor cells, they are negative for MSH (F). [Scale bars: 80 μm (C) and 20 μm (D–F).]

Fig. 5.

Schematic representation of the anterior pituitary histogenesis. Anterior pituitary develops from the epithelial primordium (Rathke's pouch), influenced by the contact with ventral diencephalon and regulated by a complex morphogenetic field of growth factors/morphogens. Around embryonic day 11, presumptive adult stem cells arise in the primordium of the anterior pituitary. They reside mostly in the perilumenal area and, although normally quiescent, possess high proliferative potential and the ability to generate all terminally differentiated cell types in response to specific physiological stimuli. During postnatal development, they contribute to all of the terminally differentiated endocrine cell types of the anterior pituitary. As a result, the adult anterior pituitary gland became a mosaic of terminally differentiated cells of different origin and with a different life history.