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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: Finally, we show that eLLRCs are contained within primary and secondary mammospheres.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.

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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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.
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Fig6: 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.

Mentions: Our data are consistent with the possibility that eLLRCs act as long-term stem/progenitor cells in the adult mammary gland. Two critical features of mammary stem/progenitor cells are that they give rise to all the cells within the gland and self-renew. The traditional assessment of these properties is the ability to repopulate the mammary gland in serial transplantation experiments. Unfortunately, the detection method for EdU requires fixation and permeabilization of cells for label visualization precluding their use in subsequent transplantation studies. Instead, to facilitate functional studies of eLLRCs, we performed in vitro MaSC assays [27,28]. An alternative test of mammary ‘stemness’ is the ability to give rise to mammospheres containing both epithelial lineages when cells are cultured under non-adherent conditions. The assay is based on the premise that, under these culture conditions, the vast majority of differentiated cells dye by anoikis, but that stem/progenitor cells can survive and give rise to mammospheres, which are, themselves, enriched in mammary stem/prognitor cells. Given that eLLRCs were enriched in the basal population, we harvested MECs from eight-week-old mice that had been labeled with EdU during embryogenesis and isolated the CD24+CD49fhigh basal cell fraction using FACS as previously described. However, we could not label for EdU without killing the cells, so we plated the sorted MaSC population at low density in 1% methylcellulose to inhibit cell aggregation. These conditions have previously been shown to ensure that each mammosphere is derived from a single cell [28]. Once formed, primary mammospheres were fixed, paraffin-embedded and analyzed for EdU labeling in serial sections, or they were dissociated and passaged in culture to form secondary mammospheres, which were also analyzed for EdU labeling after seven days. As shown in Figure 6, EdU-labeled cells were present in both primary and secondary mammospheres. Given the label dilution and rarity of cells in secondary mammospheres, further propagation of the LLRCs in third generation mammospheres was not evaluated. Importantly, in mammospheres with EdU+ cells, both K14 and K18 positive cells were detected. These results are consistent with the possibility that an individual eLLRC can give rise to an EdU-positive mammosphere and daughter cells of both luminal and basal lineages. However, since we could not sort for individual EdU-positive cells before plating, our results are not definitive. Nonetheless, the presence of EdU-positive cells within second-generation mammospheres suggests that the original eLLRCs were able to self renew during the formation of the primary mammospheres and give rise to subsequent mammospheres upon disaggregation and replating.Figure 6


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)

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

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308878&req=5

Fig6: 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.
Mentions: Our data are consistent with the possibility that eLLRCs act as long-term stem/progenitor cells in the adult mammary gland. Two critical features of mammary stem/progenitor cells are that they give rise to all the cells within the gland and self-renew. The traditional assessment of these properties is the ability to repopulate the mammary gland in serial transplantation experiments. Unfortunately, the detection method for EdU requires fixation and permeabilization of cells for label visualization precluding their use in subsequent transplantation studies. Instead, to facilitate functional studies of eLLRCs, we performed in vitro MaSC assays [27,28]. An alternative test of mammary ‘stemness’ is the ability to give rise to mammospheres containing both epithelial lineages when cells are cultured under non-adherent conditions. The assay is based on the premise that, under these culture conditions, the vast majority of differentiated cells dye by anoikis, but that stem/progenitor cells can survive and give rise to mammospheres, which are, themselves, enriched in mammary stem/prognitor cells. Given that eLLRCs were enriched in the basal population, we harvested MECs from eight-week-old mice that had been labeled with EdU during embryogenesis and isolated the CD24+CD49fhigh basal cell fraction using FACS as previously described. However, we could not label for EdU without killing the cells, so we plated the sorted MaSC population at low density in 1% methylcellulose to inhibit cell aggregation. These conditions have previously been shown to ensure that each mammosphere is derived from a single cell [28]. Once formed, primary mammospheres were fixed, paraffin-embedded and analyzed for EdU labeling in serial sections, or they were dissociated and passaged in culture to form secondary mammospheres, which were also analyzed for EdU labeling after seven days. As shown in Figure 6, EdU-labeled cells were present in both primary and secondary mammospheres. Given the label dilution and rarity of cells in secondary mammospheres, further propagation of the LLRCs in third generation mammospheres was not evaluated. Importantly, in mammospheres with EdU+ cells, both K14 and K18 positive cells were detected. These results are consistent with the possibility that an individual eLLRC can give rise to an EdU-positive mammosphere and daughter cells of both luminal and basal lineages. However, since we could not sort for individual EdU-positive cells before plating, our results are not definitive. Nonetheless, the presence of EdU-positive cells within second-generation mammospheres suggests that the original eLLRCs were able to self renew during the formation of the primary mammospheres and give rise to subsequent mammospheres upon disaggregation and replating.Figure 6

Bottom Line: Finally, we show that eLLRCs are contained within primary and secondary mammospheres.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.

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