Previously, we characterized a parity-induced mammary epithelial cell population that possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of ␤-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 whether 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 postinvolution. This approach allowed for the growth of mammary tissue from the injected cells and transient activation of the whey acidic protein promoter-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.transdifferentiation ͉ transplantation ͉ niche ͉ stem cell S omatic stem cells are maintained and regulated by their surrounding microenvironment (niche). A tissue-specific niche is a restricted locale that supports self-renewing division of stem cells and prevents them from differentiating. A simple stem cell niche has three components: localized signaling cells and extracellular matrix-controlling stem cell behavior, a specified range of signaling, and stem cell(s) (1). Any portion of the mouse mammary gland can regenerate an entire functional gland upon transplantation into a cleared mammary fat pad (2-4). This capacity remains undiminished regardless of the donor's age or reproductive history (5). Therefore, mammary stem cells are stably maintained within specific microenvironments throughout the gland for life. Mammary regeneration also occurs when dispersed epithelial cells from the glands are transplanted, suggesting that complete mammary epithelial stem cell niches may be reconstituted de novo (6-8). Stepwise (limiting) dilution of dispersed mammary cells results in a reduction of the percentage of inoculated fat pads positive for mammary epithelial growth, implying reduction in the number of mammary epithelial stem cells (7, 9). We hypothesized that the remaining cells comprised the epithelial signaling components and might support glandular regeneration if supplied with an extraneous source of stem/progenitor cells when injected into the mammary stroma. To test this hypothesis, we iso...

transplantation ͉ niche ͉ plasticity ͉ trans-differentiation ͉ regeneration S omatic stem cells are defined by how they function physiologically in the presence of heterologous cells, that is, the microenvironment or stem cell niche that balances protecting stem cells from exhaustion and protecting the host from unregulated stem cell growth. The dominance of the niche over the stem cell's autonomous phenotype has been demonstrated in several reports involving cells crossing lineage ''boundaries'' to regenerate ''foreign'' tissues. We previously demonstrated that cells isolated from the seminiferous tubules of the mature testis, when mixed together with normal mammary epithelial cells (MECs), would cooperate with these cells and contribute robust numbers of epithelial progeny to normally growing mammary glands in transplanted mammary fat pads (1). In these experiments, the testicular cells included Ϸ10% germinal stem cells (spermatogonia ␣ and ␤), Ϸ20% Sertoli cells, and the remainder primary and secondary spermatocytes and other cells at various stages of spermatogenic cell differentiation. Interstitial and vascular tissue was separated from the seminiferous tubules before dissociation. In these experiments, we were unable to distinguish whether all or just a subpopulation of the isolated seminiferous cells contributed progeny to the mammary epithelial outgrowths.In the present study, to more directly demonstrate the activity of multipotent cells (i.e., cells with the capacity to differentiate into multiple cell types) in this experimental model, we opted to begin with carefully characterized and stable multipotent stem cell populations that could be maintained indefinitely in vitro and could differentiate into multiple cell types in culture after the addition of serum factors and the removal of culture components responsible for stem cell maintenance. Toward this end, neural stem cells (NSCs) from fetal and adult WAP-Cre/Rosa26R mice were isolated and propagated in serum-free medium. Subsequently, these NSCs were mixed with equal numbers of MECs and inoculated immediately into epithelium-divested mammary fat pads of prepubertal female mice. Both fetal and adult NSCs were shown to interact with MECs on transplantation and to robustly contribute mammary epithelial-specific progeny to normal mammary outgrowths.

The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When mouse mammary tumor virus (MMTV)-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium- free mammary fat pads, they were redirected to noncarcinogenic cell fates by interaction with untransformed MECs during regenerative growth. In the presence of nontransformed MECs (50:1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu- transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells show the capacity to differentiate into normal mammary cell types, including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals, combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.

We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression in the mammary glands of parous females. PI-MEC act as lobule-limited epithelial stem/progenitor cells. To determine whether parity is necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone (Prl), or a combination of these lactogenic hormones (IHPrl). Insulin alone activated the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only I, H and Prl together were able to induce secretory differentiation and milk protein synthesis. In addition, EGF, IGF-2 and IGF-1 added individually produced activated (lacZ(+)) PI-MEC in explant cultures. Neither estrogen nor progesterone induced WAP-Cre expression in the explants. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium, indicating the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ(+) cells from virgin explants proliferated and contributed progeny to mammospheres in vitro and to epithelial outgrowths in vivo after transplantation. LacZ(+) cells induced in virgin mouse mammary explants were multipotent (like PI-MEC) in impregnated hosts producing lacZ(+) mammary alveolar structures comprised of both myoepithelial and luminal progeny. These data demonstrate PI-MEC, a mammary epithelial sub-population of lobule-limited progenitor cells, are present in nulliparous female mice before parity and, like the PI-MEC observed following parity, are capable of proliferation, self-renewal and the capacity to produce progeny of diverse epithelial cell fates.

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the capacities to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG −/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.

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Introduction Int6 has been shown to be an interactive participant with the protein translation initiation complex eIF3, the COP9 signalosome and the regulatory lid of the 26S proteasome. Insertion of mouse mammary tumor virus into the Int6 locus creates a C-terminally truncated form of the protein.Expression of the truncated form of Int6 (Int6sh) in stably transfected human and mouse mammary epithelial cell lines leads to cellular transformation. In addition, decreased expression of Int6/eIF3e is observed in approximately one third of all human breast carcinomas.