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Embryonic cells contribute directly to the quiescent stem cell population in the adult mouse mammary gland.

Boras-Granic K, Dann P, Wysolmerski JJ - Breast Cancer Res. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Section of Endocrinology and Metabolism Department of Internal Medicine, Yale University School of Medicine TAC S131, Box 208020, New Haven, CT, 06520-8020, USA. granickata@gmail.com.

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.

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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.
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Fig2: 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.

Mentions: Given that cells in the embryonic mammary buds co-express markers of both epithelial lineages (Figure 1) and in light of the ability of the mammary bud to generate a functional mammary gland when transplanted, we postulated that these structures contain mammary stem/progenitor cells [33]. Furthermore, we also reasoned that these stem/progenitor cells not only proliferate to support the outgrowth of the embryonic gland but that some proportion of these cells become quiescent to contribute to subsequent phases of development during the life cycle of the mammary gland. In order to identify quiescent or slowly cycling embryonic-derived stem/progenitor cells, we used pulse–chase labeling with the thymidine analog, EdU. Pregnant mice were pulsed twice daily with EdU from day 14.5 through day 18 in order to label dividing cells within the embryonic mammary anlage at the initiation of ductal outgrowth (Figure 2A). Mice were subsequently allowed to deliver and the mammary glands of female offspring were analyzed by immunostaining and flow cytometry to identify EdU-labeled cells.Figure 2


Embryonic cells contribute directly to the quiescent stem cell population in the adult mouse mammary gland.

Boras-Granic K, Dann P, Wysolmerski JJ - Breast Cancer Res. (2014)

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.
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Fig2: 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.
Mentions: Given that cells in the embryonic mammary buds co-express markers of both epithelial lineages (Figure 1) and in light of the ability of the mammary bud to generate a functional mammary gland when transplanted, we postulated that these structures contain mammary stem/progenitor cells [33]. Furthermore, we also reasoned that these stem/progenitor cells not only proliferate to support the outgrowth of the embryonic gland but that some proportion of these cells become quiescent to contribute to subsequent phases of development during the life cycle of the mammary gland. In order to identify quiescent or slowly cycling embryonic-derived stem/progenitor cells, we used pulse–chase labeling with the thymidine analog, EdU. Pregnant mice were pulsed twice daily with EdU from day 14.5 through day 18 in order to label dividing cells within the embryonic mammary anlage at the initiation of ductal outgrowth (Figure 2A). Mice were subsequently allowed to deliver and the mammary glands of female offspring were analyzed by immunostaining and flow cytometry to identify EdU-labeled cells.Figure 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Section of Endocrinology and Metabolism Department of Internal Medicine, Yale University School of Medicine TAC S131, Box 208020, New Haven, CT, 06520-8020, USA. granickata@gmail.com.

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.

Show MeSH
Related in: MedlinePlus