Stem and progenitor cell division kinetics during postnatal mouse mammary gland development - PubMed (original) (raw)

Stem and progenitor cell division kinetics during postnatal mouse mammary gland development

Rajshekhar R Giraddi et al. Nat Commun. 2015.

Abstract

The cycling properties of mammary stem and progenitor cells is not well understood. To determine the division properties of these cells, we administered synthetic nucleosides for varying periods of time to mice at different stages of postnatal development and monitored the rate of uptake of these nucleosides in the different mammary cell compartments. Here we show that most cell division in the adult virgin gland is restricted to the oestrogen receptor-expressing luminal cell lineage. Our data also demonstrate that the oestrogen receptor-expressing, milk and basal cell subpopulations have telomere lengths and cell division kinetics that are not compatible with these cells being hierarchically organized; instead, our data indicate that in the adult homeostatic gland, each cell type is largely maintained by its own restricted progenitors. We also observe that transplantable stem cells are largely quiescent during oestrus, but are cycling during dioestrus when progesterone levels are high.

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Conflict of interest statement

John Stingl is a paid consultant for StemCell Technologies Inc. All other authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Mammary epithelial cell population changes during postnatal development.

(a) Representative examples of whole mount carmine alum staining of mammary glands collected at 3, 4.5, 6 and 10 weeks of age in virgin C57Bl6/J mice. White dashes outline the epithelial content. (b,c) Representative flow cytometry analysis of mammary epithelial cells throughout development. (b) Expression of EpCAM and CD49f in live, lin− populations. Dot plots showing the luminal (blue) and basal (red) epithelial compartments. (c) Gating strategy of luminal cells, from B, shows further subdivision using Sca1 and CD49b expression. Three populations can be detected: Sca1+CD49b− (purple), Sca1+CD49b+ (green) and Sca1−CD49b+ (red). (d,e) Absolute number of epithelial cell subpopulations in the inguinal mammary glands of 3- to 10-week-old C57Bl6/J mice. Mean (±s.e.m) of four independent experiments for each developmental stage for data presented in a–e. ***P<0.001, as determined by analysis of variance followed by a Dunnett's multiple comparison test.

Figure 2

Figure 2. Cell division in the mammary gland during postnatal development.

(a) Quantification of Ki-67 staining among basal, ER− luminal and ER+ luminal cells in intact mammary glands of 3- to 10-week-old C57Bl6/J mice. Mean±s.e.m of four independent samples for each developmental stage. Between 10 and 35 mammary ducts were scored for each sample in the different developmental stages. (b) Representative image of sorted mammary epithelial cells stained by immunofluorescence for BrdU. Scale bar, 10 μm. A total of 12 independent samples were analysed. (c) Distribution of different mammary epithelial cell subpopulations that incorporated BrdU in the inguinal mammary glands of 3- to 10-week-old C57Bl6/J mice. Mean±s.e.m of three independent samples for each developmental stage. (d) Immunostaining of representative mammary glands from adult (≥10 weeks) C57Bl6/J mice showing the presence of Ki-67+ER−CD49b− NCL cells (arrowheads) and Ki-67+ER−CD49b+ progenitors (arrow). Scale bars, 20 μm. A total of four independent mice were analysed.

Figure 3

Figure 3. Epithelial cell turnover in the adult mammary gland.

(a) Loss of BrdU− cells over time among basal (blue), Sca1− progenitor (red), Sca1+ progenitor (green) and NCL (purple) subpopulations. BrdU was administered continuously to 24 adult, 10-week-old mice for up to 8 weeks, and 3 mice were analysed at each timepoint. (b) Absolute number of different mammary epithelial cell subpopulations in the inguinal mammary glands of adult (≥10 weeks) C57Bl6/J mice in different stages of the oestrus cycle. Mean±s.e.m of five independent mice per oestrus stage. ***P<0.001, **P<0.01, *P<0.05 as determined by analysis of variance followed by a Tukey's multiple comparisons test. (c) Protocol: adult (≥10-week-old) mice in pro-oestrus were treated with BrdU continuously (blue arrow) before culling for analysis (purple arrow), or until dioestrus where BrdU was replaced with water (green arrow) until the following pro-oestrus where mice were analysed (purple arrow). (d) Staining of BrdU-treated mammary glands. Arrows indicate clusters of BrdU+ cells. Representative images from a pool of total four independent samples. Scale bars, 30 μm. The right panels in c are close-ups of the respective areas marked with a white rectangle. Scale bars, 16 μm. (e) Representative images of BrdU staining in mammary glands of mice treated with BrdU:water during one complete oestrus cycle. Very few BrdU+ cells were detected. A total of four independent mice were analysed. Scale bars, 30 μm, right panels are close-ups of rectangle. Scale bars, 16 μm. (f) Immunostaining of representative mammary glands from adult (≥10-week-old) mice treated continuously with BrdU, showing BrdU+ER+ cells (arrows), and stained with K5 to indicate the basal compartment. Scale bars, 20 μm. A total of four independent mice were analysed. (g) Representative flow cytometry analysis of adult (≥10-week-old) C57Bl6/J mice treated continuously with EdU from pro-oestrus to metoestrus. A total of four independent mice were analysed.

Figure 4

Figure 4. Cell proliferation during the oestrus cycle.

(a) Protocol: adult (≥10-week-old) C57Bl6/J mice were administered CldU during pro-oestrus/early oestrus (blue arrows) and then IdU at metoestrus (red arrows) to detect sequential proliferation within the mammary gland. Immunofluorescent staining of adult mammary glands stained with CldU and IdU (b), and with ER, Aldh1a3 or K5 (c). Arrows indicate CldU+IdU+ cells, which demonstrate sequential cell division among mammary epithelial cells during the oestrus cycle. Representative image from four independent samples. Scale bars, 20 μm. (d) Representative flow cytometry dot plot showing the incorporation of EdU by NCL cells following injection of either oestrogen and progesterone or oil. A total of six independent adult (≥10-week-old) mice were analysed (four injected with oestrogen and progesterone, two injected with oil only). (e) NCL cells from adult (≥10-week-old) mice that are either in G0/G1 versus S/G2/M stages of the cell cycle were analysed by quantitative RT–PCR for ERα mRNA expression relative to Gapdh mRNA. Expression is relative to that of NCL G0/G1 cells. Mean±s.e.m of five independent mice.

Figure 5

Figure 5. Telomere length and telomerase activity among mammary epithelial cell subpopulations.

(a) Average telomere (Tel) intensity (a.u.) of the different sorted mammary epithelial cell populations in seven different adult (≥10-week-old) mice. Between 1,200 and 8,100 telometric spots were counted for each subpopulation. Error bars indicate s.d. All subpopulations within individual mice have telomere lengths that are significantly (***P<0.001, analysis of variance (ANOVA) followed by a Dunnett's multiple comparison test) different from telomere lengths of NCL cells, except where indicated (ns). (b) Quantification of telomerase activity in adult (≥10-week-old) C57Bl6/J mice in the different sorted populations. Telomerase activity is shown as amole of product min−1 ng−1 total cellular protein. Error bars indicate s.e.m. A total of seven independent experiments were analysed. A one-way ANOVA followed by a Dunnett's multiple comparison test was carried out and no statistical differences were observed.

References

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