In the last report, we characterized a parity-induced mammary epithelial cell population, which possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of β-galactosidase (LacZ) as a result of mammary-specific activation of a reporter gene through cre-lox recombination during pregnancy. We used this experimental model to determine if testicular cells would alter their cell fate upon interaction with the mammary gland microenvironment during pregnancy, lactation and involution. Adult testicular cells, isolated from seminiferous tubules, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. The host mice were bred 6-8 weeks later and examined 20-30 days post involution. This approach allowed for the growth of mammary tissue from the injected cells, transient activation of the WAP (whey acidic protein)-Cre gene during pregnancy and lactation leading to Cre-lox recombination and constitutive expression of LacZ from its promoter. Here we show that cells from adult seminiferous tubules interact with mammary epithelial cells during regeneration of the gland. They adopt mammary epithelial progenitor cell properties including self-renewal and the production of cell progeny, which differentiate into functional mammary epithelial cells. Our results provide evidence for the ascendancy of the tissue microenvironment over the intrinsic nature of cells from an alternative adult tissue. A fundamental issue in stem cell biology is whether adult somatic stem cells are capable of accessing alternate tissue sites and continue functioning as stem cells in the new microenvironment. To address this issue relative to neurogenic stem cells in the mouse mammary gland microenvironment, we mixed wild-type mammary epithelial cells with bona fide neural stem cells (NSC) isolated from WAP-Cre/R26R mice and inoculated them into cleared fat pads of immune-compromised females. Hosts were bred 6-8 weeks later and examined post involution. This allowed for mammary tissue growth, transient activation of the WAP-Cre gene, recombination and constitutive expression of LacZ. The NSC and their progeny contributed to mammary epithelial growth during ductal morphogenesis and the Rosa26-LacZ reporter was activated by WAP-Cre expression during pregnancy. NSC-derived, LacZ-positive cells expressed mammary-specific functions including milk protein synthesis while others adopted myoepithelial cell fates. Thus, NSC and their progeny enter mammary epithelium-specific niches and adopt the function of similarly endowed mammary cells. This result supports the conclusion that tissue specific signals emanating from the stroma and from the differentiated somatic cells of the mouse mammary gland can redirect the neural stem cell to produce cellular progeny committed to mammary epithelial cell fates. Our studies have identified a lobule-limited, pluripotent mammary epithelial progenitor that is identified in post-parous mammary gland by the conditional activation of β-galactosidase (βgal) from a ubiquitous promoter (Rosa26) via whey acidic protein promoter (WAP)-driven Cre-lox recombination. Spermatogenic and neural stem cells, carrying these reporters, are re-directed to mammary epithelial progenitor cell fates in the presence of wild type mammary epithelial cells during regenerative mammary outgrowth in epithelium-cleared fat pads. Surviving βgal + cells in post-lactation, involuted glands indicates progeny from the non-mammary donor. Testicular and neural stem cells arise from the embryonal ectoderm. To test whether mesoderm-derived stem cells could respond to mammary epithelial-specific signals and adopt ectodermal cell fates, we mixed FACS-purified Lin-, cKit+ adult male bone marrow cells with equal numbers of mammary epithelial cells and implanted the mixture into cleared mammary fat pads of immuno-compromised female hosts. Following pregnancy, lactation and involution in the recipient females, the resulting outgrowths were examined for βgal-expressing cells. Robust contribution of βgal + epithelial progeny from bone marrow donor cells was observed in the resulting chimeras. FISH for the Y chromosome in mammary cells demonstrated the male donor origin of the βgal+ cells. Co-expression of mammary epithelial-specific markers and βgal demonstrated that the non-mammary donor cell progeny had adopted bona fide mammary epithelial cell phenotypes. The βgal + cells were found both in luminal and basal mammary epithelial cell types and the former were shown to be capable of milk protein synthesis in pregnant recipients. The bone marrow-derived progeny exhibited properties consistent with those previously demonstrated for parity-identified lobule-limited epithelial progenitor cells. The male βgal + cells were capable of symmetric expansion and retention of their pluripotent nature over three serial passages. Our evidence shows that the signals provided by the mammary microenvironment are capable of redirecting mesoderm-derived adult bone marrow-derived cells to produce mammary epithelial cell progeny. Long-lived stem/progenitor cells in somatic tissues are hypothesized to protect themselves from mutation and cancer risk through a process of selective segregation of their template DNA strands during asymmetric division. Mouse mammary epithelium contains label-retaining epithelial cells (LREC) that divide asymmetrically and retain their template DNA strands. During pregnancy the mammary epithelial compartment undergoes extreme proliferation and differentiation facilitated by stem/progenitor cells. Methods- Immunohistochemistry and autoradiography was performed on murine mammary glands that had been labeled with 5-bromodeoxyuridine (5BrdU) during allometric ductal growth to investigate the co-expression of DNA label-retention and estrogen receptor-α (ERα) or progesterone receptor (PR) during pregnancy. A second DNA label ([3H]-thymidine) was administered during pregnancy to identify label-retaining cells (LRC), which subsequently enter the cell cycle. Using the same methods, we investigated the co-localization of 5BrdU with smooth muscle actin, CD31, cytokeratin and desmin in periductal or peri-acinar LRC in mammary tissue from pregnant mice subsequent to a long chase period to identify label-retaining cells. ERα-positive (ERα+) and PR-positive (PR+) cells represented roughly 30-40% of the LREC, which is <1.0% of the epithelial subpopulation. Pregnancy altered the percent of LREC expressing ERα. LRC situated in periductal or peri-acinar positions throughout the gland do not express epithelial, endothelial or myoepithelial markers and these undifferentiated LRC persist throughout pregnancy. Additionally, new LREC are created during alveologenesis and LRC found in other tissue types (e.g. endothelium and nerve) within the mammary fat pad become double-labeled during pregnancy, an indication that they may also divide asymmetrically. Our results support the premise that there is a subpopulation of LREC in the mouse mammary gland that persists during alveologenesis. These cells react to the hormonal cues during pregnancy and enter the cell cycle while continuing to retain, selectively, their original template DNA. In addition, non-epithelial LRC are found in periductal or peri-acinar positio [summary truncated at 7800 characters]
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