EGF controls the in vivo developmental potential of a mammary epithelial cell line possessing progenitor properties

The bilayered mammary epithelium comprises a luminal layer of secretory cells and a basal layer of myoepithelial cells. Numerous data suggest the existence of self-renewing, pluripotent mammary stem cells; however, their molecular characteristics and differentiation pathways are largely unknown. BC44 mammary epithelial cells in culture, display phenotypic characteristics of basal epithelium, i.e., express basal cytokeratins 5 and 14 and P-cadherin, but no smooth muscle markers. In vivo, after injection into the cleared mammary fat pad, these cells gave rise to bilayered, hollow, alveolus-like structures comprising basal cells expressing cytokeratin 5 and luminal cells positive for cytokeratin 8 and secreting beta-casein in a polarized manner into the lumen. The persistent stimulation of EGF receptor signaling pathway in BC44 cells in culture resulted in the loss of the in vivo morphogenetic potential and led to the induction of active MMP2, thereby triggering cell scattering and motility on laminin 5. These data (a) suggest that BC44 cells are capable of asymmetric division for self-renewal and the generation of a differentiated progeny restricted to the luminal lineage; (b) clarify the function of EGF in the control of the BC44 cell phenotypic plasticity; and (c) suggest a role for this phenomenon in the mammary gland development.     

Derivation of myoepithelial progenitor cells from bipotent mammary stem/progenitor cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5(+)/K19(-) type, and a K5(+)/K19(+) type. Under specific culture conditions, bipotent K5(+)/K19(-) stem/progenitor cells differentiated into stable clonal populations that were K5(-)/K19(-) and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5(+)/K19(-) cells which are bipotent. These K5(-)/K19(-) cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease.     

Caveolin-1-Deficient Mice Have An Increased Mammary Stem Cell Population with Upregulation of Wnt/?-Catenin Signaling

Here, we show that a caveolin-1 (Cav-1) deficiency leads to an amplification of the adult mammary stem cell population, both in vivo and in vitro. First, the expression of two stem cell markers, Sca-1 and Keratin 6, is dramatically increased in the hyperplastic mammary ducts of Cav-1 deficient mice, suggesting that loss of Cav-1 induces the accumulation of a progenitor cell population in the mammary gland. To independently validate these results, we reconstituted mammary acini formation in vitro via a 3D Matrigel assay system--using primary cultures of mammary epithelial cells derived from WT and Cav-1 deficient mice. We show that Cav-1 null 3D epithelial structures display an intense increase in the expression of three stem cell markers, i.e., Sca-1, keratin 6 and keratin 5. Overall, we observed a 2-to-3 fold increase in the number of Cav-1 KO acini that are positive for a given stem cell marker. Also, we show that such amplification of progenitor cells has functional consequences, as demonstrated by the abnormal presence of myoepithelial cells in the hyperplastic lesions of Cav-1 deficient mammary glands. Finally, we provide evidence that hyper-activation of Wnt/?-catenin signaling may constitute one of the down-stream mechanisms leading to mammary stem cell accumulation. The longevity and slow-dividing properties of mammary stem cells facilitates the accumulation of genetic alterations, and renders these progenitor cells the likely precursors of malignant derivatives. As such, we propose that loss of Cav-1 induces the accumulation of mammary stem cells, and that this event may be an initiating factor during mammary tumorigenesis.     

Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.     

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for regeneration of tissues that can potentially replace/repair diseased and damaged tissue through differentiation into epithelial, myoepithelial and/or cuboidal/columnar cells in the udder with minimal risk of rejection and side effects.     

Notch signaling regulates mammary stem cell function and luminal cell-fate commitment

The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.     

Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland

The role of estrogen in promoting mammary stem cell proliferation remains controversial. It is unclear if estrogen receptor (ER)-expressing cells have stem/progenitor activity themselves or if they act in a paracrine fashion to stimulate stem cell proliferation. We have used flow cytometry to prospectively isolate mouse mammary ER-expressing epithelial cells and shown, using analysis of gene expression patterns and cell type-specific markers, that they form a distinct luminal epithelial cell subpopulation that expresses not only the ER but also the progesterone and prolactin receptors. Furthermore, we have used an in vivo functional transplantation assay to directly demonstrate that the ER-expressing luminal epithelial subpopulation contains little in vivo stem cell activity. Rather, the mammary stem cell activity is found within the basal mammary epithelial cell population. Therefore, ER-expressing cells of the mammary epithelium are distinct from the mammary stem cell population, and the effects of estrogen on mammary stem cells are likely to be mediated indirectly. These results are important for our understanding of cellular responses to hormonal stimulation in the normal breast and in breast cancer.     

Isolation and characterization of functional mammary gland stem cells

Abstract.  Significant advances in the stem-cell biology of several tissues, including the mammary gland, have occurred over the past several years. Recent progress on stem-cell fate determination, molecular markers, signalling pathways and niche interactions in haematopoietic, neuronal and muscle tissue may provide parallel insight into the biology of mammary epithelial stem cells. Taking advantage of approaches similar to those employed to isolate and characterize haematopoietic and epidermal stem cells, we have identified a mammary epithelial cell population with several stem/progenitor cell qualities. In this article, we review some recent data on mammary epithelial stem/progenitor cells in genetically engineered mouse models. We also discuss several potential molecular markers, including stem-cell antigen-1 (Sca-1), which may be useful for both the isolation of functional mammary epithelial stem/progenitor cells and the analysis of tumour aetiology and phenotype in genetically engineered mouse models. In different transgenic mammary tumour models, Sca-1 expression levels, as well as several other putative markers of progenitors including keratin-6, possess dramatically altered expression profiles. These data suggest that the heterogeneity of mouse models of breast cancer may partially reflect the selection or expansion of different progenitors.     

Growth and differentiation of progenitor/stem cells derived from the human mammary gland

Estrogen is necessary for the full development of the mammary gland and it is also involved in breast cancer development. We set out to identify and characterise progenitor/stem cells in the human mammary gland and to explore the role of estrogen in their proliferation and differentiation. Three candidate stem cell populations were isolated: double positive (DP) cells co-expressed the luminal and myoepithelial markers, EMA and CALLA, respectively, whereas double negative (DN) cells did not express these cell surface markers; side population (SP) cells were characterised by their differential ability to efflux the dye Hoechst 33342. The ABC transporter, breast cancer resistance protein (BCRP) was more highly expressed in SP cells than in non-SP cells and a specific BCRP inhibitor, Ko143, reduced SP formation, suggesting that BCRP confers the SP phenotype in mammary epithelial cells, as has been demonstrated in other tissues. Interestingly, SP cells were double negative for the EMA and CALLA antigens and therefore represent a separate and distinct population to DP cells. Single cell multiplex RT-PCR indicated that the SP and DN cells do not express detectable levels of ERalpha or ERbeta, suggesting that estrogen is not involved in their proliferation. DP cells expressed ERalpha but at a lower level than differentiated luminal cells. These findings invoke a potential strategy for the breast stem/progenitor cells to ignore the mitogenic effects of estrogen. All three cell populations generated mixed colonies containing both luminal and myoepithelial cells from a single cell and therefore represent candidate multipotent stem cells. However, DN cells predominately generated luminal colonies and exhibited a much higher cloning efficiency than differentiated luminal cells. Further characterisation of these candidate progenitor/stem cells should contribute to a better understanding of normal mammary gland development and breast tumorigenesis.     

Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations

The stem cell niche comprises stem cells (SCs), stromal cells, soluble factors, extracellular matrix constituents and vascular networks. The ability to identify signals that regulate SC self-renewal and differentiation is confounded by the difficulty in isolating pure SC niche components in sufficient quantities to enable their biochemical characterisation. Here, we report the extracellular (secretome) and adherent plasma membrane proteomes of three distinct epithelial cell subpopulations isolated and immortalized from the mouse mammary gland--basal and mammary stem cell (basal/MaSC), luminal progenitor (LP) and mature luminal (ML) cell lines. GeLC-MS/MS-based proteomic profiling revealed a distinct switch in components modulating Wnt and ephrin signalling, and integrin-mediated interactions amongst the three cell subpopulations. For example, expression of ephrin B2, ephrin receptors A1, and A2, as well as integrins α2β1 and α6β4 were shown to be enriched in basal/MaSCs, relative to LP and ML cells. Conspicuously, Wnt10a was uniquely detected in basal/MaSCs, and may modulate the canonical Wnt signalling pathway to maintain basal/MaSC activity. By contrast, non-canonical Wnt signalling might be elevated in ML cells, as evidenced by the high expression levels of Wnt5a, Wnt5b, and the transmembrane tyrosine kinase Ror2.     

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.     

Differentiation of separated mouse mammary luminal epithelial and myoepithelial cells cultured on EHS matrix analyzed by indirect immunofluorescence of cytoskeletal antigens.

We have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cell-cell and cell-matrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cell-cell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. (J Histochem Cytochem 47:1513-1524, 1999)     

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERα^neg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.     

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1⁺) and basal/myoepithelial (CD10⁺) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Matrix compliance and RhoA direct the differentiation of mammary progenitor cells

The regenerative capacity of the mammary gland following post-lactational involution depends on the presence of multipotent stem or progenitor cells. Mammary progenitor cells exist as a quiescent and self-renewing population capable of differentiating into luminal epithelial and myoepithelial cells and generating ductal and alveolar structures. The fate choices of these cells are regulated by several soluble signals as well as their surrounding extracellular matrix. Whereas matrix stiffness has been implicated in organ-specific differentiation of embryonic and mesenchymal stem cells, the effects of substratum compliance on the more limited fate switches typical of tissue-specific progenitor cells are unknown. Here, we examined how the mechanical properties of the microenvironment affect the differentiation of mammary progenitor cells. Immortalized human mammary progenitor cells were cultured on synthetic hydrogels of varying stiffness, and their self-renewal and fate decisions were quantified. We found that cells cultured on soft substrata differentiated preferentially into luminal epithelial cells, whereas those cultured on stiff substrata differentiated preferentially into myoepithelial cells. Furthermore, pharmacological manipulations of cytoskeletal tension in conjunction with analysis of gene expression revealed that mechanical properties of the microenvironment signal through the small GTPase RhoA and cytoskeletal contractility to modulate the differentiation of mammary progenitor cells. These data suggest that subtle variations in the mechanical compliance of a tissue can direct the fate decisions of its resident progenitor cells.     

Characterization of stem cells from the murine adult mammary gland

The postnatal mammary morphogenesis comprises two steps, first, formation of a system of branching ducts at puberty and second, alveologenesis during pregnancy. The mammary epithelium is organized as a bilayer, composed of two cellular types, basal myoepithelial and luminal epithelial. The remarkable regenerative properties revealed in serial transplantation experiments suggest that the adult mammary epithelium harbors stem cells. Various strategies including analysis of DNA label-retaining cells, transgenic approach, and in vivo transplantation assay, have been used to isolate and characterize murine mammary stem and progenitor cells. Their molecular characteristics remain to be defined precisely but notable progress have been already made in the enrichment and identification of these cells. Current studies favor the hypothesis of a basal-type mammary stem cells expressing high levels of alpha 6, beta1 and beta 3 integrin chains, the major receptors of extracellular matrix proteins. Luminal-type progenitors may participate in the establishment of the bilayered alveolar epithelium during pregnancy.