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. 2013 Oct 3;13(4):419-32.
doi: 10.1016/j.stem.2013.07.006.

Mobilized adult pituitary stem cells contribute to endocrine regeneration in response to physiological demand

Karine Rizzoti  1 , Haruhiko Akiyama, Robin Lovell-Badge
Affiliations

Mobilized adult pituitary stem cells contribute to endocrine regeneration in response to physiological demand

Karine Rizzoti et al. Cell Stem Cell. .

Abstract

Pituitary hormone deficiencies, with Growth Hormone deficiency being most frequent (1 in 3,500-10,000 births), cause significant morbidity. Regeneration of missing endocrine cells would be a significant improvement over hormone replacement therapies, which incur side effects and do not mimic physiological secretion patterns. Recent in vitro studies have identified a population of adult pituitary progenitors that express the HMG box transcription factors SOX2 and SOX9. Here, we apply cell-lineage tracing analysis to demonstrate that SOX2- and SOX9-expressing progenitors can self-renew and give rise to endocrine cells in vivo, suggesting that they are tissue stem cells. Moreover, we show that they can become mobilized and differentiate into the appropriate endocrine cell types in response to physiological stress. Our results highlight the pituitary as a model for exploring how physiological changes influence stem cell behavior and suggest that manipulation of endogenous pituitary stem cells is a potential therapeutic strategy for pituitary deficiencies.

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Figures

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Graphical abstract
Figure 1
Figure 1
SOX2- and SOX9-Positive Cells Are the Sphere-Forming Cells (A and B) SOX2-EGFP and SOX9-IRES-GFP alleles faithfully recapitulate SOX2 (A) and SOX9 (B) expression in the pituitary. Note that SOX2-EGFP is not present in cells where SOX2 staining is cytoplasmic (arrows). (C and D) FAC sorting of 3 week-old Sox2EGFP/+ and 4-week-old Sox9iresGFP/iresGFP. (E and F) Spheres are observed in SOX2-EGFP and SOX9-IRES-GFP-positive fractions (bright field and live fluorescence). (G and H) Spheres obtained from SOX2-positive cells (G, DAPI and immunofluorescence for GFP and anterior pituitary hormones) and SOX9-positive cells (H, DAPI and immunofluorescence for GFP and all anterior pituitary hormones) are able to give rise to hormonal cells in differentiating conditions. Scale bars: 50 μm for (A) and (B) and 5 μm for (G) and (H). See also Figure S1.
Figure 2
Figure 2
SOX2- and SOX9-Positive Progenitors Give Rise to Endocrine Cells in the Embryo and to SOX2;SOX9-Double-Positive Putative Progenitors in the Adult (A–F) Sox2CreERT2 embryonic lineage tracing. (G–L) Sox9ires-CreERT2 embryonic lineage tracing. 4-OHT (0.1 to 0.15 mg/g/day) was given to pregnant females at 11 and 12.5 dpc. Pituitaries were harvested at 18.5 dpc and immunofluorescence was performed for GFP, SOX2, SOX9, and anterior pituitary cell lineages (Pit-1 for GH, PRL, and TSH-secreting cells and their progenitors, ACTH for corticotrophs, and LH and FSH for gonadotrophs). (L) Double-positive cells were counted for each lineage in at least three embryos. “Other” refers to the proportion of EYFP+ve cells that were not identified. (M) Proportions of SOX+ve progenitors stained at 18.5 dpc after induction of Sox2CreERT2 early, before SOX9 is expressed (11.5+12.5), or late, as SOX9 is upregulated (13.5 dpc), along with Sox9ires-CreERT2 results. Data are presented as mean ± SEM. (N) Model recapitulating results from the embryonic lineage tracings according to expression timing of SOX2, SOX9, and Cre induction. (O and P) Fetal SOX2- (O) and SOX9- (P) positive progenitors give rise to adult progenitors. 4-OHT (0.1 to 0.15 mg/g) was given to pregnant females at 11.5 (Sox9ires-CreERT2) and 13.5 dpc (Sox2CreERT2). Pituitaries were harvested postnatally at 8 weeks. EYFP expression was observed in particular in SOX2/9-double-positive progenitors. Insets show SOX2 and SOX9 staining in triple-positive cells. Scale bars: 10 μm for all panels. See also Table S1.
Figure 3
Figure 3
SOX2- and SOX9-Positive Postnatal Progenitors Give Rise to Endocrine Cells and Folliculo-Stellate Cells (A–F) Sox2CreERT2 postnatal lineage tracing. (G–L) Sox9ires-CreERT2 postnatal lineage tracing. 4-OHT (1 mg) was given to P0–P2 pups and pituitaries were harvested at 4 weeks. (A–L) Immunofluorescence for EYFP, SOX2, SOX9, and anterior pituitary hormones were then performed. Double-positive cells were counted for each lineage in at least four animals. “Other” refers to the proportion of EYFP+ve cells that were not identified. SOX2 and SOX9 progenitors participate in pituitary growth postnatally and give rise to differentiated cells in all endocrine lineages. (M–O) Sox9ires-CreERT2 adult lineage tracing. Tamoxifen (5 mg/25 g/day for 3 days) was given to 8 week-old animals. Pituitaries were harvested at one year (M and N) or after 2 months (O). (M) Triple SOX2;SOX9;EYFP and double (N) GH;EYFP and (O) S100;EYFP are shown. Insets show SOX2 and SOX9 staining in triple-positive cells. Scale bars: 10 μm for all panels. See also Figure S3 and Table S1.
Figure 4
Figure 4
Estrogen Treatment in Males Induces Proliferation of Adult SOX2-Positive Cells In Vivo and Indirectly Induces Formation of More Pituispheres In Vitro E2 pellets (0.2 mg) were implanted in 8-week-old males. BrdU was then injected for 3 days and pituitaries were harvested. (A) Double immunofluorescence in a sham and E2-treated animals for BrdU and SOX2. There is more BrdU in the E2-treated sample and SOX2;BrdU-double-positive cells are numerous (arrows). (B) Percentage of BrdU-positive nuclei in sham versus treated animals. There is a significant increase of BrdU incorporation in E2-treated animals (p = 0.003). (C) The percentage of SOX2-positive cells in the anterior pituitary is not significantly affected by E2 treatment. (D) In contrast there is a significant increase in the percentage of SOX2;BrdU-double-positive/SOX2+ve cells in E2-treated animals (p = 0.0003). (E) The percentage of caspase-3-positive cells is not affected. (F) In vivo E2 treatment increases significantly the number of pituispheres obtained after dissociation (p = 0.006). (G) In contrast, E2 treatment in vitro, after pituitary dissociation, does not affect the number of pituisphere formed. E2 effect on SOX2-positive cells is therefore likely to be indirect. (H) In agreement with this hypothesis, double immunofluorescence for SOX2 and ERα/β show almost exclusive expression patterns in the anterior pituitary. Scale bars: 50 μm. Data are presented as mean ± SEM.
Figure 5
Figure 5
Pituitary Target Organ Ablation Induces Proliferation of Adult SOX2-Positive Cells In Vivo and Formation of More Pituispheres In Vitro Gonadectomies (A–D) and adrenalectomies (E) were performed in 8-week-old males. BrdU was then injected for 5 days and pituitaries were harvested (A–D). (A) Percentage of BrdU-positive nuclei in sham versus gonadectomized animals. There is a significant increase of BrdU incorporation in gonadectomized animals (p = 0.02). (B) The percentage of SOX2-positive cells in the anterior pituitary is not significantly affected by the ablation. (C) In contrast there is a significant increase in the percentage of BrdU;SOX2-double-positive/SOX2+ve cells in gonadectomized animals (p = 0.001). Pituitaries from gonadectomized (D, p = 0.0006) or adrenalectomized (E, p = 0.005) animals give rise to significantly more spheres in vitro than their sham counterparts. Data are presented as mean ± SEM.
Figure 6
Figure 6
Pituitary Target Organ Ablation Induces Differentiation of Adult SOX9-Positive Stem Cells Adrenalectomies were performed 24 hr after the last tamoxifen administration (5 mg/25 g/day for 5 days). Pituitaries were harvested 1 week later. (A) ACTH-positive corticotrophs were counted on two sections/animal and the surface area of the sections was measured. We observed significantly more corticotrophs after adrenal ablation (p = 0.023). (B) We almost exclusively observe EYFP;ACTH-double-positive corticotrophs after adrenalectomies in Sox9ires-CreERT2/+;R26REYFP/+ animals (adrenalectomy n = 5, 2 females and 3 males, sham-operated n = 5, 3 females and 2 males, p = 0.0005, with no difference found between males and females). (C) Double immunofluorescence illustrating the differentiation of EYFP+ve SOX9 progenitors in ACTH+ve corticotrophs in a Sox9ires-CreERT2/+;R26REYFP/+ animal after adrenalectomy. Scale bar: 10 μm. (D) Proportion of new corticotrophs generated from SOX9+ve progenitors. We first assessed the efficiency of SOX9-IRES-CreERT2 by counting the number of EYFP;SOX9-double-positive cells 48 hr after 5 days of tamoxifen treatment. Eighteen percent (SD = 3.2, n = 3) of SOX9+ve cells were EYFP+ve. We then measured the surface area of all the sections where we counted ACTH;EYFP-double-positive cells in adrenalectomized animals (n = 5). We corrected this number so that it represented 100% and not just the 18% of cells generated from SOX9+ve cells. This amounted to 21 corticotrophs/mm2, representing 19% of the new corticotrophs. Data are presented as mean ± SEM. See also Figure S4 and Table S1.
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