Corinne A. Boulanger

Parity-induced mouse mammary epithelial cells are pluripotent, self-renewing and sensitive to TGF-β1 expression

A parity-induced mammary population, marked by β-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-β1 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-β1-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-TGF-β1 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.

Interaction with the mammary microenvironment redirects spermatogenic cell fate in vivo

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 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 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.

The mammary microenvironment alters the differentiation repertoire of neural stem cells

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 (MECs) with bona fide neural stem cells (NSCs) isolated from WAP-Cre/Rosa26R mice and inoculated them into cleared fat pads of immunocompromised females. Hosts were bred 6–8 weeks later and examined postinvolution. This allowed for mammary tissue growth, transient activation of the WAP-Cre gene, recombination, and constitutive expression of LacZ. The NSCs and their progeny contributed to mammary epithelial growth during ductal morphogenesis, and the Rosa26-LacZ reporter gene was activated by WAP-Cre expression during pregnancy. Some NSC-derived LacZ+ cells expressed mammary-specific functions, including milk protein synthesis, whereas others adopted myoepithelial cell fates. Thus, NSCs 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 NSCs to produce cellular progeny committed to MEC fates.

Reducing mammary cancer risk through premature stem cell senescence

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-TGFβ1, we discovered that mammary epithelial stem cells were prematurely aged due to ectopic expression of TGF-β1. 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/N×WAP-TGF-β1 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-β1 females while 15 were collected from 29 wild type sisters. Premalignant mammary epithelial cells in infected glands were identified by transplantation of single cell (1×105) suspensions into nulliparous hosts and testing for hyperplastic outgrowth. Although the number of positive takes was significantly reduced with TGF-β1 cells, both MMTV-infected TGF-β1 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.

Reprogramming human cancer cells in the mouse mammary gland.

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 normal mammary microenvironment suppresses the tumorigenic phenotype of mouse mammary tumor virus-neu-transformed mammary tumor cells

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 non-carcinogenic cell fates by interaction with untransformed MECs during regenerative growth. In the presence of non-transformed 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.

Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation.

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. J. Cell. Physiol. 212:729–736, 2007.

Mammary epithelial stem cells: transplantation and self-renewal analysis

Abstract.  An entire mammary epithelial outgrowth, capable of full secretory differentiation, may be comprised of the progeny of a single cellular antecedent. This conclusion is based upon the maintenance of retroviral insertion sites within the somatic DNA of successive transplant generations derived from a single mammary fragment. In addition, dissociation of these clonal dominant glands and implantation of dispersed cells at limiting dilution demonstrated that both duct‐limited and lobule‐limited outgrowths were developed, as well as complete, fully differentiated glands. Thus, transplantation has revealed three distinct mammary epithelial progenitors in the mouse. Similar studies have extended this observation to rat mammary tissue. Recently, using cre‐lox conditional activation of reporter genes, a new epithelial progenitor, specific for mammary secretory epithelium in postlactation females has been uncovered. In situ, these cells were shown to regenerate secretory lobules upon successive pregnancies. In transplant studies, they demonstrated the capacity for self‐renewal and contributed to the new generation of all of the known epithelial cell types among mammary epithelium. In limiting dilution, the parity‐induced progenitors were capable of engendering lobule‐limited and duct‐limited outgrowths in their entirety, but not completely developed glands. Serial transplant studies indicate that these progenitors have a significant but limited capacity for self‐renewal.

Mammary stem cell repertoire: new insights in aging epithelial populations

The proliferative lifespan of mammary stem cells was examined in serially transplanted clonal-dominant epithelial populations. Five successive transplant generations were done. The epithelial cell number in each outgrowth expands approximately 500-fold in nulliparous hosts and approximately 10000-fold in impregnated hosts. Despite this, all resulting mammary outgrowths showed lineal identity with the original. Growth senescence was observed in some implants beginning at the third generation in impregnated recipients. The ability of an individual implant to support ductal morphogenesis and also secretory lobule development decayed at independent rates. Individual implants from a single clonal-dominant outgrowth occasionally gave rise to markedly different ductal development within the same host indicating an epithelial cell autonomous mechanism in ductal patterning. Both premalignant and malignant populations appeared focally within the aging transplants. These populations were also lineally related to the original outgrowth supporting the conclusion that the primary growth was derived clonally from one or a few lineally related antecedents. The premalignant and malignant descendant populations no longer exhibit growth senescence suggesting that they are supported by a perpetually self-renewing progenitor. Our evidence indicates that a single mammary cell may have the capacity to self-renew through five transplant generations. Even some sixth generation implants show vigorous growth.

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

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 beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability 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 CDbetageo 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.