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

Kata Boras-Granic Pamela Dann John J Wysolmerski 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. - Introduction In mice, mammary gland development begins around embryonic day 10.5 (e10.5) with the formation of bilateral mammary lines between the fore and hind limb buds along the ventral-lateral borders of the embryo. Cells within the mammary line coalesce into five distinct pairs of placodes (three thoracic and two inguinal). Over the next several days, each mammary placode expands and invaginates into the underlying mesenchyme to form a mammary bud (Figure 1A). Mammary rudiments have very low proliferative activity between e11.25 and e13.5 and the initial phases of mammary development are thought to rely on cell migration from the epidermis rather than proliferation of mammary epithelial cells [1-3]. Active proliferation within the mammary epithelium begins at e14.5 [4]. By e15.5, the distal end of the mammary bud begins to elongate into the underlying dermal mesenchyme to form a sprout. The sprout grows downward into the mammary fat pad, an adipocyte-rich 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 m. e, embryonic day; MG, mammary gland; WT, wild type. stromal compartment and begins to branch, forming the rudimentary ductal tree by e18.5. By birth, the mammary epithelium consists of a primary duct and about 10 to 15 branches located within the proximal end of the nascent mammary fat pad. The postnatal mammary gland continues to undergo periodic development and remodeling. The nascent ductal system grows isometrically until puberty when ductal morphogenesis accelerates in response to hormonal cues. At puberty, terminal end buds (TEBs) form at the tips of mammary ducts [5]. TEBs are the predominant sites of epithelial proliferation during puberty, as the ducts invade the surrounding stromal tissue and elongate to the distal end of the fat pad. In virgin animals, cyclical development and regression of small ductules and alveolar buds occurs along the epithelial ducts with each estrous cycle. During pregnancy, these buds further develop into fully formed alveoli that fill the fat pad in preparation for milk production during lactation [5-7]. This process involves vigorous proliferation and the secretory differentiation of a large number of new epithelial cells. Once lactation ceases, the alveoli regress during involution, most of the newly generated epithelial cells die and the mammary gland returns to a resting state. The cycle of pregnancy, lactation, and involution can repeat itself multiple times during the reproductive lifespan of an animal, suggesting the presence of stem/progenitor cells to supply each new cycle of expansion. By definition, tissue stem cells have the capacity to generate all cell types of the tissue in which they reside and are able to self-renew in order to support long-term homeostasis of an organ. The existence of a population of stem cells in the adult mammary gland was initially shown by the ability of any fragment of the mammary gland to reconstitute an entire gland upon transplantation as well as the reproduction of genetic chimerism upon transplantation of fragments of a chimeric gland [8,9]. More recently, the existence of individual stem cells was demonstrated by the ability of a single cell to regenerate the entire mammary epithelium upon transplantation [10,11]. These experiments implicate the existence of a multi-potent cell that can give rise to myoepithelial, luminal and alveolar lineages. However, recent lineage tracing studies disagree on whether multipotent cells actually give rise to the various mammary epithelial lineages during development and reproductive cycles in vivo, or whether lineage-restricted progenitor cells are induced to behave in a multi-potent manner in transplantation studies [12-14]. One potential source of multi-potent mammary stem cells may be the embryonic mammary bud [15]. Intact mammary buds from as early as e12 can give rise to an entire mammary gland when transplanted into the cleared fat pad of pubertal mice. In contrast, when dissociated into single cells and grafted into cleared fat pads, the repopulating potential of the embryonic mammary epithelium was (...truncated)