Embryonic cells contribute directly to the quiescent stem cell population in the adult mouse mammary gland - PubMed (original) (raw)
Embryonic cells contribute directly to the quiescent stem cell population in the adult mouse mammary gland
Kata Boras-Granic et al. Breast Cancer Res. 2014.
Abstract
Introduction: Studies have identified multi-potent stem cells in the adult mammary gland. More recent studies have suggested that the embryonic mammary gland may also contain stem/progenitor cells that contribute to initial ductal development. We were interested in determining whether embryonic cells might also directly contribute to long-lived stem cells that support homeostasis and development in the adult mammary gland.
Methods: We used DNA-label retention to detect long label-retaining cells in the mammary gland. Mouse embryos were labeled with 5-ethynl-2'-deoxyuridine (EdU) between embryonic day 14.5 and embryonic day 18.5 and were subsequently sacrificed and examined for EdU retention at various intervals after birth. EdU retaining cells were co-stained for various lineage markers and identified after fluorescence activated cell sorting analysis of specific epithelial subsets. EdU-labeled mice were subjected to subsequent 5-bromo-2'-deoxyuridine administration to determine whether EdU-labeled cells could re-enter the cell cycle. Finally, EdU-labeled cells were grown under non-adherent conditions to assess their ability to form mammospheres.
Results: We demonstrate embryonically-derived, long label-retaining cells (eLLRCs) in the adult mammary gland. eLLRCs stain for basal markers and are enriched within the mammary stem cell population identified by cell sorting. eLLRCs are restricted to the primary ducts near the nipple region. Interestingly, long label retaining cells (labeled during puberty) are found just in front of the eLLRCs, near where the ends of the ducts had been at the time of DNA labeling in early puberty. A subset of eLLRCs becomes mitotically active during periods of mammary growth and in response to ovarian hormones. Finally, we show that eLLRCs are contained within primary and secondary mammospheres.
Conclusions: Our findings suggest that a subset of proliferating embryonic cells subsequently becomes quiescent and contributes to the pool of long-lived mammary stem cells in the adult. eLLRCs can re-enter the cell cycle, produce both mammary lineages and self-renew. Thus, our studies have identified a putative stem/progenitor cell population of embryonic origin. Further study of these cells will contribute to an understanding of how quiescent stem cells are generated during development and how fetal exposures may alter future breast cancer risk in adults.
Figures
Figure 1
Luminal and myoepithelial lineage marker expression during embryonic mammary gland development. (A) Schematic representation of embryonic mammary rudiment formation in the female from e11.5 to birth. Five pairs of mammary glands form in the female mouse. (B) Immunostaining for K14 (green) and Gata3 (red) (top), K14 (green) and p63 (red) (middle), and K14 (green) and K8 (red) (bottom) in WT MG at e11, e13, e15, and newborn (one day old). Scale bars, 40 μm. e, embryonic day; MG, mammary gland; WT, wild type.
Figure 2
Embryonically EdU-labeled cells persist in the mammary ducts of adult mice. (A) Schematic protocol to study embryonic label retention in the mouse mammary gland. (B) EdU label incorporation was examined in paraffin sections from e15 embryos labeled for 24 hours. EdU+ cells are located in the epithelial bud and surrounding mesenchyme. Paraffin sections stained for EdU from MGs of newborn (C), three-week-old (D), and eight-week-old (E) mice after EdU administration between e14.5 and e18. L = lumen. (F) Percentage of EdU-labeled cells at various time-points after embryonic administration. (n = 3 to 5 mice/stage) (G) Diagram of LLRC location in eight week MG. (H) Percentage of EdU+ cells that express basal or luminal lineage markers in eight week MGs. (n = 1,000 cells). Bars represent SE. [See Additional file 2]. (I) Paraffin section of proximal portion of 10-week chased MG co-stained for EdU, K14 and Gata3. Edu + cells (red) are located predominantly in the basal layer of the ducts and are a subpopulation of the K14-positive cells (white; see yellow arrows). EdU+ cells are also located in the luminal layer of the ducts and are a rare subpopulation of the Gata3-positive cells (green; see white asterisks). (J-M) Summary of flow cytometry data. (J) MGs were isolated from eight-week-old EdU pulse-chased mice and analyzed by flow cytometry for expression of cell surface markers, CD24 and CD49f. EdU+ cells represent 1.2% of total mammary epithelial cells Lin-CD24+ [See Additional file 1]. (K) Immunofluorescence analysis for EdU+ cells in cytospins of sorted Lin-CD24+CD49flow luminal and Lin-CD24+CD49fhigh basal cells. (L) Representative histogram FACS plot of sorted and fixed populations of Lin-CD24+CD49flow luminal and Lin-CD24+CD49fhigh basal cells. (M) Average percentage of EdU+ cells in Lin-CD24+CD49flo luminal (2.2%) and Lin-CD24+CD49fhigh basal (8.88%) cell populations. Data represent mean ± SEM of three independent experiments. Scale bars, 20 μm, mm = mammary mesenchyme. e, embryonic day; EdU, 5-ethynl-2′-deoxyuridine; FACS, fluorescence activated cell sorting; LLRC, long label retaining cells; MG, mammary gland; SE, standard error; SEM, standard error of the mean.
Figure 3
Embryonic EdU labeling followed by pubertal BrdU labeling demonstrates overlapping but distinct LLRC populations. (A) Scheme for sequential embryonic labeling with EdU from e14.5 to e18.5 followed by pubertal labeling with BrdU from day 21 to 25. (B) Diagrammatic representation of the relative localization of EdU-retaining and BrdU-retaining epithelial cells in the adult mammary gland at eight-weeks of age. EdU-retaining cells localized to the main ducts in the nipple region, while BrdU cells are localized between the EdU-positive cells and the lymph node. (C) Percentage of EdU-positive and BrdU-positive cells at different time points following labeling. Representative sections co-stained for BrdU and the myoepithelial markers K14 (D) or p63 (E), or the luminal markers, estrogen receptor (F) or progesterone receptor (G). Boxes represent areas of inset. Arrows in insets point to double-labeled cells. (H) Percentage of BrdU-positive cells that also stain for K14, p63, ER or PR. BrdU, 5-bromo-2′-deoxyuridine; e, embryonic day; EdU, 5-ethynl-2′-deoxyuridine; ER, estrogen receptor; LLRC, long label retaining cells.
Figure 4
A subset of eLLRCs re-enter the cell cycle during postnatal development and tissue remodeling. Embryonically EdU-labeled (e14.5 to e18) animals were administered BrdU for five days at different stages of postnatal development. Paraffin sections of mammary glands at (A) five days (newborn), (B) three weeks and five days (puberty), (C) mid-pregnancy (12.5 days) and (D) four months (virgin-nulliparous) were co-stained for EdU and BrdU. K14 staining is included in some samples to aid in tissue structure visualization. Double-labeled EdU + BrdU+ cells (see white arrows) were evident in glands analyzed at all time points. In addition, a cohort of female mice that received a second BrdU pulse at puberty or pregnancy was also chased for an additional five to seven weeks (E,F). Examination of BrdU pulse-chased mammary glands demonstrated double-labeled LRCs at the end of puberty (eight weeks of age) (E), and occasional rare EdU + BrdU + LRCs at the end of involution (F). White arrows denote double-labeled cells. Scale bar, 20 μm. Rectangles highlight areas shown at higher magnification. BrdU, 5-bromo-2′-deoxyuridine; e, embryonic day; EdU, 5-ethynl-2′-deoxyuridine; eLLRCs, embryonically derived long label retaining cells; LRCs, label retaining cells.
Figure 5
Hormone-dependent activation of a subset of eLLRCs in the adult mammary gland. (A) Diagrammatic representation of protocol used to examine proliferation of embryonic LLRCs during hormone treatment in adult virgin mice. Embryonically EdU-labeled adult mice were injected daily for two weeks with estrogen and progesterone (E + P) or vehicle and with BrdU twice/day. (B) FACS profiles of luminal CD24+CD49flow and basal CD24+CD49fhigh cell populations isolated from either control or E + P treated 10-week old mammary glands. (C , D) Immunofluorescent staining of paraffin sections for EdU and BrdU. Scale bar represent 20 μm. (E) Histograms showing quantification of the eLLRCs that proliferate after hormone treatments. Hormone treatment resulted in a 1.7-fold increase in the number of EdU+ LLRCs labeled with BrdU. Data represent mean ± SEM of n = 3 mice each. BrdU, 5-bromo-2′-deoxyuridine; EdU, 5-ethynl-2′-deoxyuridine; eLLRCs, embryonically derived long label retaining cells; SEM, standard error of the mean.
Figure 6
eLLRCs from the MaSC population generate multi-lineage mammospheres. (A) Three-dimensional image of mammosphere stained for EdU (red), K14 (white) and K18 (green). Representative bright field images of gross morphology of primary (B) and secondary (D) mammospheres grown under ultra low non-adherent conditions in vitro. (C , E) Immunofluorescence analysis of paraffin sections of mammospheres stained for EdU (red) with nuclear counterstain, DAPI (blue). C represents three different primary mammospheres while E is a second-generation mammosphere. DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynl-2′-deoxyuridine; eLLRCs, embryonically derived long label retaining cells; MaSC, mammary stem cell.
References
- Lee MY, Racine V, Jagadpramana P, Sun L, Yu W, Du T, Spencer-Dene B, Rubin N, Le L, Ndiaye D, Bellusci S, Kratochwil K, Veltmaat JM. Ectodermal influx and cell hypertrophy provide early growth for all murine mammary rudiments, and are differentially regulated among them by Gli3. PLoS One. 2011;6:e26242. doi: 10.1371/journal.pone.0026242. - DOI - PMC - PubMed
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