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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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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.
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Fig3: 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.

Mentions: Given that embryonically EdU-labeled cells were restricted to the proximal portion of the mammary gland near the nipple region, it suggested to us that quiescent eLLRCs might be deposited in the ducts behind the active growth front at the time of labeling. In order to test this idea, we performed sequential labeling first with EdU from e14.5 to e18 and then with BrDU for five days beginning at the initiation of puberty at three weeks of age (21 days) (Figure 3A). As depicted in Figure 3B, at the end of the BrDU pulse period (25 days), approximately 77% of epithelial cells were BrdU-positive based on immunofluorescence analysis. We then examined the mammary glands for EdU and BrdU labeling at eight weeks of age, five weeks after BrdU labeling. The percentage of BrdU-positive cells decreased to 6.5%. In contrast to the EdU label-retaining cells, the BrdU label-retaining cells were enriched in the luminal rather than the basal compartment. Twenty percent of BrdU-positive cells expressed p63 and 14% expressed K14 while one third were positive for the luminal hormone receptors, PR and ER (Figure 3E). These results are in agreement with previously published data identifying adult LLRCs [18,19,21]. Strikingly, BrdU label-retaining cells were enriched in the proximal portion of the mammary gland just before the lymph node (Figure 3B). While there was some overlap with the EdU-positive cells, the BrdU-positive cells were mostly between the EdU-positive cells and the lymph node, approximately at the location where the ends of the ducts had been at the time that the BrDU pulse was given. These data demonstrate that a subset of cells near the active growth front can become LLRCs suggesting that quiescent stem/progenitor cells are deposited along the elongating ducts as a function of active morphogenesis occurring at the leading edge.Figure 3


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig3: 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.
Mentions: Given that embryonically EdU-labeled cells were restricted to the proximal portion of the mammary gland near the nipple region, it suggested to us that quiescent eLLRCs might be deposited in the ducts behind the active growth front at the time of labeling. In order to test this idea, we performed sequential labeling first with EdU from e14.5 to e18 and then with BrDU for five days beginning at the initiation of puberty at three weeks of age (21 days) (Figure 3A). As depicted in Figure 3B, at the end of the BrDU pulse period (25 days), approximately 77% of epithelial cells were BrdU-positive based on immunofluorescence analysis. We then examined the mammary glands for EdU and BrdU labeling at eight weeks of age, five weeks after BrdU labeling. The percentage of BrdU-positive cells decreased to 6.5%. In contrast to the EdU label-retaining cells, the BrdU label-retaining cells were enriched in the luminal rather than the basal compartment. Twenty percent of BrdU-positive cells expressed p63 and 14% expressed K14 while one third were positive for the luminal hormone receptors, PR and ER (Figure 3E). These results are in agreement with previously published data identifying adult LLRCs [18,19,21]. Strikingly, BrdU label-retaining cells were enriched in the proximal portion of the mammary gland just before the lymph node (Figure 3B). While there was some overlap with the EdU-positive cells, the BrdU-positive cells were mostly between the EdU-positive cells and the lymph node, approximately at the location where the ends of the ducts had been at the time that the BrDU pulse was given. These data demonstrate that a subset of cells near the active growth front can become LLRCs suggesting that quiescent stem/progenitor cells are deposited along the elongating ducts as a function of active morphogenesis occurring at the leading edge.Figure 3

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