A parity-induced mammary population, marked by b-galactosidase expression conditionally activated through cre-lox recombinase originates in WAP-Cre/Rosa-lox-STOP-lox-LacZ (WAP-Cre/Rosa-LacZ) female mice during pregnancy, lactation and involution. During subsequent pregnancies, these parity-induced mammary epithelial cells (PI-MEC) proliferated to produce new secretory acini composed of secretory luminal cells and myoepithelium. In serial transplantation assays, PI-MEC were able to self-renew over several transplant generations and to contribute significantly to the resulting mammary outgrowths. In limiting dilution transplantation, they proliferated to produce both luminal and myoepithelial cells, comprised both lobule-limited and duct-limited epithelial outgrowths, and differentiated into all the cellular subtypes recognized in murine mammary epithelium. TGF-b1 expression from the whey acidic protein promoter (WAP) in triply transgenic females did not prevent the appearance of PI-MEC after pregnancy despite the absence of full lactation or their ability to proliferate and produce progeny with diverse cellular fates in situ upon subsequent pregnancies. However, in transplants from triple transgenic parous females, the WAP-TGF-b1-positive PI-MEC did not contribute to the newly recapitulated mammary outgrowths, suggesting that they were incapable of expansive cellular proliferation (self-renewal). This result is consistent with our earlier publication that WAP-TGFb1 expression in mammary epithelium induces premature stem cell senescence in mammary transplants and decreases mammary cancer risk in mouse mammary tumor virus (MMTV)-infected females even after multiple pregnancies.

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 tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we showed that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. Fluorescence in situ hybridization confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric DNA, as well as immunochemistry for human-specific breast epithelial cytokeratins and human-specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66- to 660-fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results show that human embryonal carcinoma–derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.

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.

The reproductive capacity of the mammary epithelial stem cell is reduced coincident with the number of symmetric divisions it must perform. In a study of FVB/ N mice with the transgene, WAP-TGFb1, we discovered that mammary epithelial stem cells were prematurely aged due to ectopic expression of TGF-b1. To test whether premature aging of mammary epithelial stem cells would have an impact on susceptibility or resistance to mammary cancer, female littermates from FVB/ N6WAP-TGF-b1 mating were injected with mouse mammary tumor virus (MMTV) at 8 ± 10 weeks of age. A total of 44 females were inoculated, maintained as breeders and observed for tumor development for up to 18 months. Only one mammary tumor appeared in 17 TGF-b1 females while 15 were collected from 29 wild type sisters. Premalignant mammary epithelial cells in infected glands were identi®ed by transplantation of single cell (1610 5 ) suspensions into nulliparous hosts and testing for hyperplastic outgrowth. Although the number of positive takes was signi®cantly reduced with TGF-b1 cells, both MMTV-infected TGF-b1 and wild type cells produced hyperplastic outgrowths suggesting that premalignant transformation was achieved in each group. The results suggest a positive correlation between the procreative life-span of mammary epithelial stem cells and mammary cancer risk. Oncogene (2001) 20, 2264 ± 2272.

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.

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