Human milk protein production in xenografts of genetically engineered bovine mammary epithelial stem cells

Background

In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow''s milk using gene transfer.

Methods and Findings

We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.

Conclusions

These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.     

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.     

Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay

Methods to identify and enumerate primitive, and typically rare, undifferentiated cells in normal tissue using functional endpoints are powerful tools for acquiring insights into the mechanisms that regulate normal tissue stem cell turnover and differentiation. In this paper, we describe a xenotransplantation-based protocol that allows mammary stem cells with in vivo tissue regenerative properties to be specifically detected and quantified among the heterogeneous cell populations obtained from dissociated normal human mammary tissue. This methodology involves implanting a collagen gel containing the test cells in combination with supportive fibroblasts under the kidney capsule of highly immune-deficient, hormone-supplemented mice and then, 4 weeks later, searching for regenerated human cells with in vitro clonogenic activity. Quantification of the input human mammary stem cells is achieved using standard limiting dilution transplant approaches. This approach circumvents the need to modify the mouse mammary fat pad, and is objective, rapid (～5 weeks) and economical to perform.     

Proliferation and differentiation in the human breast during pregnancy

Using multiple immunofluorescence labelling on human breast tissues obtained and freshly frozen at the 12th, 15th, and 18th weeks of pregnancy, we have shown that markers of mammary functional differentiation, milk proteins (beta-casein and kappa-casein), are synthesised by actively cycling (Ki67 positive) as well as non-cycling (Ki67 negative) cells. These results demonstrate that functional differentiation/maturation does not coincide with loss of proliferative potential in human mammary luminal epithelial cells. In addition, we have examined expression patterns of integrin subunits (alpha1, alpha2, alpha3, alpha6, beta1, and beta4) and extracellular matrix components (laminin, fibronectin, collagen I, and collagen IV), since they have been shown to exert influences on mammary differentiation and morphogenesis in vitro. Compared to human breast tissues obtained from non-pregnant women, a decrease in alpha2 labelling on luminal epithelial cells was observed, particularly in expanding acini that showed abundant Ki67 positivity. The expression patterns of other integrin subunits, however, did not change, indicating that the expression patterns of most integrins existing prior to pregnancy are sufficient to support the morphological and functional development associated with milk protein synthesis.     

The biology of human breast epithelial progenitors

Current evidence suggests that similar to other tissues in the human body mammary epithelia cells are being maintained by the unique properties of stem cells, undifferentiated as well as lineage-restricted progenitors. Because of their longevity, proliferation and differentiation potentials these primitive breast epithelial cells are likely targets of transforming mutations that can cause them to act as cancer initiating cells. In this context, understanding the molecular mechanisms that regulate the normal functions of the human breast epithelial stem cells and progenitors and how alterations to these same mechanisms can confer a cancer stem cell phenotype on these rare cell populations is crucial to the development of new and more effective therapies again breast cancer. This review article will examine the current state of knowledge about the isolation and characterization of human breast epithelial progenitors and their relevance to breast cancer research.     

Concurrent expression of basal and luminal epithelial markers in cultures of normal human breast analyzed using monoclonal antibodies

The aim of this study was to investigate the retention in culture of the antigens characteristic of the two mammary epithelial subclasses, basal and luminal epithelium. Primary and secondary cultures of normal human mammary-gland cells were used for immunolocalization experiments with monoclonal antibodies to luminal and basal epithelium. In contrast to the in vivo situation, in which reactivity was only seen in basal cells that were negative for the luminal antigen, we found the homogeneous expression of the basal marker by all of the cultured cells at second passage, and the simultaneous expression of the luminal marker by some of these cells. Characterization of the basal antigen expressed in culture using sodium-dodecyl-sulfate/polyacrylamide gel electrophoresis and immunoblotting techniques showed it to be a 51-kilodalton keratin peptide with an isoelectric pH of 5.4, and confirmed its similarity to the antigen expressed in vivo. Our findings thus demonstrated the coordinate expression of the basal and luminal antigens in cells cultured on solid substrates. The availability of monoclonal antibodies to epithelial-subclass-specific markers of the human mammary gland now makes it feasible to search for culture conditions that would allow the maintenance and manipulation of cell differentiation in vitro.     

BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.     

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.     

Mammary gland development: Role of basal myoepithelial cells

Mammary epithelium is organized as a bilayer with a layer of luminal secretory cells and a layer of basal myoepithelial cells. To dissect the specific functions of these two major compartments of the mammary epithelium in mammary morphogenesis we have used genetically modified mice carrying transgenes or conditional alleles whose expression or ablation were cell-type specific. Basal cells are located in close proximity to mammary stroma and directly interact with the extracellular matrix (basement membrane) during all their lifespan. On the contrary, luminal secretory cells during early stages of the postnatal mammary development have only limited contacts with basement membrane and become exposed to the extracellular matrix only during late developmental stages at the end of pregnancy and in lactation. Consistently perturbation of beta1-integrin function specifically in the luminal layer of the mammary epithelium, did not interfere with mammary morphogenesis until the second part of pregnancy but led to impaired secretory differentiation and lactation. On the contrary, ablation of beta1-integrin gene in the basal mammary epithelial cells resulted in a more precocious phenotype: disorganized branching in young virgin animals and a complete arrest of lobuloalveolar development. Further, a constitutive activation of beta-catenin signaling due to expression of N-terminally truncated (stabilized) beta-catenin specifically in basal myoepithelial cells resulted in accelerated differentiation of luminal secretory cells in pregnancy, precocious postlactational involution, increased angiogenesis and development of mammary tumors. Altogether these data suggest that basal mammary epithelial cells can affect growth and differentiation of luminal secretory cells, have an impact on the epithelium-stroma relationships and, thereby, play an important role in the process of mammary morphogenesis and differentiation.     

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting

Summary Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.     

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f⁺ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further.     

NOTCH signaling in Sertoli cells regulates gonocyte fate

Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, the regulatory mechanisms that govern gonocyte maintenance and transition to SSCs are poorly understood. Recently, we reported that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence—the first suggestion of the potential role of this signaling pathway in the testis.

This Extra View will review what is known about NOTCH signaling, particularly in Sertoli cells and germ cells in the testes, by providing a background on germ cell biology and a summary of our recently published data on NOTCH1 signaling in Sertoli cells. We also describe additional data showing that aberrant proliferation and differentiation of gonocytes in response to constitutive activation of NOTCH1 signaling in Sertoli cells involves de novo expression of cell cycle proteins and a marked upregulation of the KIT receptor. These data further suggest that NOTCH signaling orchestrates a dynamic balance between maintenance and differentiation of gonocytes in the perinatal testis.     

Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.     

An in vitro model of epithelial cell growth stimulation in the rodent mammary gland

Abstract. Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFα and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro – in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.     

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.     

Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type-specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type-specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.