Mammary gland stem cells: More puzzles than explanations

Mammary gland stem cells (MaSC) have not been identified in spite of extensive research spanning over several decades. This has been primarily due to the complexity of mammary gland structure and its development, cell heterogeneity in the mammary gland and the insufficient knowledge about MaSC markers. At present, Lin (-) CD29 (i) CD49f (i) CD24 (+/mod) Sca- 1 (-) cells of the mammary gland have been reported to be enriched with MaSCs. We suggest that the inclusion of stem cell markers like Oct4, Sox2, Nanog and the mammary gland differentiation marker BRCA-1 may further narrow down the search for MaSCs. In addition, we have discussed some of the other unresolved puzzles on the mammary gland stem cells, such as their similarities and/or differences with mammary cancer stem cells, use of milk as source of mammary stem cells and the possibility of in vitro differentiation of embryonic stem (ES) cells into functional mammary gland structures in this review. Nevertheless, it is the lack of identity for a MaSC that is curtailing the advances in some of the above and other related areas.     

Identification of putative bovine mammary epithelial stem cells by their retention of labeled DNA strands

Stem cells appear to retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily injections of 5-bromo-2-deoxyuridine (BrdU). Five weeks later, BrdU-labeled mammary epithelial cells were still evident. The percentage of BrdU-labeled epithelial cells was greatest in the lower region of the mammary gland, near the gland cistern, and was decreased toward the periphery of the parenchymal region, where the ducts were invading the mammary fat pad. Increased numbers of BrdU-labeled epithelial cells in basal regions of the gland are likely a consequence of decreased proliferation rates and increased cell cycle arrest in this area. In peripheral regions of mammary parenchyma, the percentage of heavily labeled epithelial cells averaged 0.24%, a number that is consistent with estimates of the frequency of stem cells in the mouse mammary gland. Epithelial label-retaining cells seemingly represent a slowly proliferating population of cells, as 5.4% of heavily labeled cells were positive for the nuclear proliferation antigen Ki67. Because epithelial label-retaining cells contain estrogen receptor (ER)-negative and ER-positive cells, they apparently comprise a mixed population, which I suggest is composed of ER-negative stem cells and ER-positive progenitors. Continuing studies will address the usefulness of this technique to identify bovine mammary stem cells and to facilitate studies of stem cell biology.     

14-3-3σ (sigma) regulates proliferation and differentiation of multipotent p63-positive cells isolated from human breastmilk

The mammary gland is a dynamic organ that only undergoes complete differentiation during pregnancy. Differentiation is fuelled by asymmetric division of stem cells that reside in normally quiescent niches in the resting gland in response to pregnancy-associated hormones. Loss of regulation of stem cells is believed to underlie some breast cancers. This process is poorly understood in humans since it is difficult to extract stem cells from the lactating gland. We have identified a p63-positive population in breastmilk that proliferates and differentiates into at least two separate mammary lineages in culture. Nuclear translocation of p63 coincides with expression of the cell-cycle arrest protein 14-3-3σ (Sigma) and precedes differentiation. Transient down-regulation of Sigma promotes maintenance of the p63-positive population without affecting normal differentiation. We propose that p63-postive cells from breastmilk represent a novel source of cells to model regulation of mammary gland development and tumorigenesis.     

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.     

The dynamics of murine mammary stem/progenitor cells

The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups.     

Mammary development and breast cancer: the role of stem cells

The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies.     

Deciphering the Mammary Epithelial Cell Hierarchy

Clonal assays offer a powerful approach to dissecting the many events involved in the generation and maintenance of complex tissues from an undifferentiated stem cell pool. The application of such quantitative functional methodologies to studies of the hematopoietic system have been key to defining the hierarchy of progenitor subsets that reflect an irreversible stepwise process of lineage restriction. Recent studies now suggest that a similar paradigm applies to the normal mammary gland. The adult mouse mammary gland maintains a population of stem cells that generate biologically distinct and physically separable subpopulations of mammary epithelial progenitors which, in turn, generate terminally differentiated cells. Suggestive parallels between mouse and human mammary cells point to the likelihood that a similarly structured multi-step differentiation program characterizes the mammary gland from both species.     

Mammary gland stem cells: current status and future challenges

Distinct subsets of cells, including cells with stem cell-like properties, have been proposed to exist in normal human breast epithelium and breast carcinomas. The cellular origins of epithelial cells contributing to gland development, tissue homeostasis and cancer are, however, still poorly understood. The mouse is a widely used model of mammary gland development, both directly by studying the mouse mammary epithelial cells themselves and indirectly, by studying development, morphogenesis, differentiation and carcinogenesis of xenotransplanted human breast epithelium in vivo. While in early studies, human or mouse epithelium was implanted as fragments into the mouse gland, more recent technical progress has allowed the self-renewal capacity and differentiation potential of distinct cell populations or even individual cells to be interrogated. Here, we review and discuss similarities and differences between mouse and human gland development with particular emphasis on the identity and localization of stem cells, and the influence of the surrounding microenvironment. It is concluded that while recent advances in the field have contributed immense insight into how the normal mammary gland develops and is maintained, significant discrepancies exist between the mouse and human gland which should be taken into consideration in current and future models of mammary stem cell biology.     

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.     

Developmental Stage and Time Dictate the Fate of Wnt/β-Catenin-Responsive Stem Cells in the Mammary Gland

The mammary epithelium undergoes extensive growth and remodeling during pregnancy, suggesting a role for stem cells. Yet their origin, identity, and behavior in the intact tissue remain unknown. Using an Axin2(CreERT2) allele, we labeled and traced Wnt/β-catenin-responsive cells throughout mammary gland development. This reveals a switch in Wnt/β-catenin signaling around birth and shows that, depending on the developmental stage, Axin2(+)?cells contribute differently to basal and luminal epithelial cell lineages of the mammary epithelium. Moreover, an important difference exists between the developmental potential tested in transplantation assays and that displayed by the same cell population in?situ. Finally, Axin2(+) cells in the adult build alveolar structures during multiple pregnancies, demonstrating the existence of a Wnt/β-catenin-responsive adult stem cell. Our study uncovers dynamic changes in Wnt/β-catenin signaling in the mammary epithelium and offers insights into the developmental fate of mammary gland stem and progenitor cells.     

Simulating mouse mammary gland development: cell ageing and its relation to stem and progenitor activity

BACKGROUND: Somatic stem and progenitor cell division is likely to be an important determinant of tumor development. Each division is accompanied by a risk of fixing genetic mutations, and/or generating innately immortal cells that escape normal physiological controls. AIM: Using biological information, we aimed to devise a theoretical model for mammary gland development that described the effect of various stem/progenitor cells activities on the demographics of adult mammary epithelial cell populations. RESULTS: We found that mammary ductal trees should develop in juvenile mice despite widely variant levels of activity in the progenitor compartment. Sequestration (inactivation) of progenitor cells dramatically affected the aging-maturation of the population without affecting the total regenerative capacity of the gland. Our results showed that if stem and progenitor cells can be demonstrated in glands regenerated by serial transplantation, they originated in a canonical primary stem cell (providing a functional definition of mammary stem cells). Finally, when the probability of symmetric division of stem cells increased above a threshold, the mammary epithelial population overall was immortal during serial transplantation. CONCLUSIONS: This model provides, (1) a theoretical framework for testing whether the phenotypes of genetically modified mice (many of which are breast cancer models) derive from changes of stem and progenitor activity, and (2) a means to evaluate the resolving power of functional assays of regenerative capacity in mammary epithelial cell populations.     

In search of mammary gland stem cells

Summary The evidence for mammary epithelial stem cells and their phenotypic characteristics in normal and neoplastic development is reviewed. The presence of stem cells in all parts of the mammary parenchyma at all stages of differentiation has been demonstrated by transplantation experiments. The phenotypic characterization of stem cells has been defined by a battery of monospecific antibodies. These studies suggest that a mammary epithelium stem cell compartment exists in the basal layer of the gland as well as in the end bud. Whether these same stem cells are expressed in mammary preneoplasias and neoplasias has not been answered conclusively. Phenotypic markers specific for stem cells and stably expressed in transformed cell populations are needed to follow the fate of stem cells.Dedicated to Professor Stuart Patton on the occassion of his 70th birthday.     

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.     

β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands

Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed β-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the β-casein promoter, independent of β-casein expression.     

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

On murine mammary epithelial stem cells: discovery, function, and current status

Cell-surface markers expressed on mammary stem cells and progenitors have helped to establish a preliminary mammary cell lineage hierarchy. Further characterization of these cells depends on overcoming several technical obstacles.Remarkable progress has been made in the past decade in the isolation and characterization of mouse mammary stem cells and progenitors, as nicely reviewed in the article by Visvader and Smith (2011). Following in the footsteps of the hematopoietic system and analogous to bone marrow transplantation, the mammary gland can be reconstituted following transplantation of cells into the cleared mammary fat pad (see review by Medina 2011). Taking advantage of these similarities as well as the availability of genetically engineered mice (GEM), our laboratory initially used magnetic bead and fluorescence-activated cell sorting (FACS) and SCA enhanced green fluorescent protein (EGFP) knock-in mice to identify mammary gland progenitors (Welm et al. 2002). We also attempted to identify and isolate quiescent cells using a BrdU label retention strategy that had been successfully applied in the epidermal and intestinal epithelium. Subsequently, the identification of several cell-surface markers expressed on mammary stem cells and progenitors has resulted in an explosion in the field, and helped to define a preliminary mammary cell lineage hierarchy. These studies on the normal mammary gland have also provided the basis for hypotheses into potential mechanisms accounting for the heterogeneity of breast cancer subtypes (Behbod and Rosen 2004).One intrinsic difference between the hematopoietic system and the mammary gland, however, is the requirement for tissue dissociation in the latter case to facilitate the isolation of single cells required for FACS sorting. Even when using freshly isolated cells, there is a concern that these rather lengthy dissociation protocols may alter the expression of cell-surface molecules and properties of cells following disruption of the mammary gland architecture. Even short-term cell culture of primary mammary epithelial cells may alter the expression of cell-surface molecules. At present, single gene markers of mammary stem cells have not been identified, so the application of knock-in mice, e.g., the use of LGR5-EGFP to identify intestinal stem cells and perform lineage-tracing experiments (Barker et al. 2007), has not been feasible. One alternative approach may be to use pathway reporters, as recently described by Zeng and Nusse (2010), who used an axin-lacZ knock-in mouse to identify cells with canonical Wnt signaling with increased mammary repopulating activity. We have used a similar approach in a p53-null mouse mammary cancer model following lentiviral transduction with a Wnt reporter construct to identify cells with enhanced canonical Wnt signaling. These cells displayed a significant overlap with cell-surface markers in the basal-like tumors shown to enrich for tumor-initiating cells (Zhang et al. 2010).The use of multiple pathway reporters with different fluorescent reporters may provide a new approach to complement the current dependence on cell-surface markers. Fluorescent reporters also have the potential to help precisely visualize and model the location of mammary stem cells and progenitors in situ using multiphoton and other sophisticated microscopic techniques. The ability to visualize single stem cells in their niche environment and to follow both symmetric versus asymmetric division ultimately will be required for the next advances in the field. Recent studies on the paracrine effects of the steroid hormones, estrogen and progesterone, on mammary gland stem cells and progenitors illustrate the need to understand the spatial relationships among the various epithelial and stromal cell types present in the mammary gland. These studies will need to include cells from the immune system such as macrophages, neutrophils, etc., and derivatives of mesenchymal stem cells. Hopefully, in the near future it may be feasible to reconstitute and study these interactions in vitro, but for the present time this can be studied in GEM models. In addition, there is increasing evidence for the coexistence of quiescent and active adult stem cells in mammals (Li and Clevers 2010), but these distinct populations and their spatial and temporal relationships in the mammary gland remain to be discovered. Application of single-cell analysis using newly developed microfluidic platforms has the potential to help elucidate the potential heterogeneity of signaling pathways and gene expression in mammary stem cells and progenitors. Finally, there is a critical need for lineage-tracing experiments in the normal mammary gland to validate the proposed hierarchy for stem cells and progenitors, as well as to identify the cells of origin for different subtypes of breast cancer. Comparative studies of the murine and human stem cell populations in both the normal mammary gland and different breast cancer subtypes hold enormous potential for the future. Thus, despite the remarkable progress in this field, much remains to be done.     

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue,but has a latent negative effect on cell proliferation

The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine?s effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation.     

Differentiation dynamics of mammary epithelial stem cells from Korean holstein dairy cattle under ECM-free conditions

The “stem cells” are commonly defined as “cells capable of self-renewal through replication and differentiating into specific lineages”. The mammary gland contains functional stem/progenitor cells. The current study was planned with the objectives to study the differentiation dynamics of Korean Holstein mammary epithelial stem cells (KHMESCs) under the optimum culture conditions. Lineage negative KHMESCs isolated from mammary tissue of lactating cows have shown the typical differentiation dynamics with formation of lobulo–alveolar structures in in vitro culture. This suggests the existence of bipotential mammary epithelial stem cells in the mammary gland. The strong mRNA expression of pluripotency factors indicates stemness, whereas expression of milk protein genes and epithelial cell-specific gene indicate their differentiation capabilities. Further, immunostaining results have shown the differentiation capabilities of KHMESCs into both luminal and basal lineages under the extracellular matrix (ECM, matrigel) free environment. However, under matrigel, the differentiation process was comparatively higher than without matrigel. Immunostaining results also suggested that differentiated cells could secrete milk proteins such as β-casein. To our knowledge, these data represent the first report on the differentiation dynamics and establishment of mammary epithelial stem cells from Korean Holstein with typical stemness properties. It was observed that isolated KHMESCs had normal morphology, growth pattern, differentiation ability, cytogenetic and secretory activity even without ECM. Therefore, it is concluded that established KHMESCs could be used for further studies on Korean Holstein dairy cows related to lactation studies, as non-GMO animal bioreactors and stem cell-based management of bovine mastitis including post-mastitis damage.     

Survival and engraftment of mouse embryonic stem cells in the mammary gland

Embryonic stem (ES) cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES cells to improve functional outcome following mammary gland injury. This study investigates the feasibility of implanting mouse ES cells labeled with enhanced green fluorescence protein in the developing mammary glands in order to acquire lineage-committed cells in mammary (mammary gland epithelial cell or luminal cell). Cells implanted in high numbers (5 × 10(6) cells per mammary gland) survived in the majority of the mice and nearly 38.4% of the surviving cells were CK18(+) at 15th week following the transplantation. Our results may provide a technique instrument on advanced therapy of breast diseases and the mammary regeneration after breast ablated partly.     

Mammary development in the embryo and adult: a journey of morphogenesis and commitment

Mammary gland development occurs through distinctive stages throughout embryonic and pubertal development and reproductive life. At each stage, different signals are required to induce changes in both the epithelium and the surrounding mesenchyme/stroma. Recent studies have provided new insights into the origin, specification and fate of mammary stem and progenitor cells and into how the differentiated lineages that comprise the functional mammary gland are determined. The development of new tools and culture techniques has also enabled the factors that influence branching morphogenesis in the embryonic and pubertal gland to be identified. A surprising recent discovery has been that mammary epithelial cells commit to differentiated lineages using the same signalling pathways that regulate lineage determination in T helper cells.