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.     

Reconstruction of human mammary tissues in a mouse model

Establishing a model system that more accurately recapitulates both normal and neoplastic breast epithelial development in rodents is central to studying human breast carcinogenesis. However, the inability of human breast epithelial cells to colonize mouse mammary fat pads is problematic. Considering that the human breast is a more fibrous tissue than is the adipose-rich stroma of the murine mammary gland, our group sought to bypass the effects of the rodent microenvironment through incorporation of human stromal fibroblasts. We have been successful in reproducibly recreating functionally normal breast tissues from reduction mammoplasty tissues, in what we term the human-in-mouse (HIM) model. Here we describe our relatively simple and inexpensive techniques for generating this orthotopic xenograft model. Whether the model is to be applied for understanding normal human breast development or tumorigenesis, investigators with minimal animal surgery skills, basic cell culture techniques and access to human breast tissue will be able to generate humanized mouse glands within 3 months. Clearing the mouse of its endogenous epithelium with subsequent stromal humanization takes 1 month. The subsequent implantation of co-mixed human epithelial cells and stromal cells occurs 2 weeks after humanization, so investigators should expect to observe the desired outgrowths 2 months afterward. As a whole, this model system has the potential to improve the understanding of crosstalk between tissue stroma and the epithelium as well as factors involved in breast stem cell biology tumor initiation and progression.     

Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for ‘normal’ gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.     

Reprogramming non-mammary and cancer cells in the developing mouse mammary gland

The capacity of any portion of the murine mammary gland to produce a complete functional mammary outgrowth upon transplantation to an epithelium-divested fat pad is unaffected by the age or reproductive history of the donor. Likewise, through serial transplantations, no loss of potency is detected when compared to similar transplantations of the youngest mammary tissue tested. This demonstrates that stem cell activity is maintained intact throughout the lifetime of the animal despite aging and the repeated expansion and depletion of the mammary epithelium through multiple rounds of pregnancy, lactation and involution. These facts support the contention that mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue, to produce dispersed cells results in the desecration of the protection afforded by the "niche" and leads to a reduced capacity of dispersed epithelial cells (in terms of the number transplanted) to recapitulate complete functional mammary structures. Our studies demonstrate 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 cells, including mouse and human cancer cells, may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells.     

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.     

Mammary stem cells come of age, prospectively

Two recent reports have contributed direct evidence for the existence of a pluripotent mouse mammary epithelial stem cell. In both reports, the investigators have prospectively isolated an enriched fraction of mammary stem cells using fluorescence-activated cell sorting from freshly dispersed epithelial cells. This fraction of cells, upon transplantation in limiting dilution (in some cases as a single cell), produces complete mammary development within the host mammary fat pad. These studies extend and confirm earlier work that demonstrated that retroviral-tagged mammary fragments produce complete functional mammary glands comprising their clonal progeny upon fat-pad transplantation. This technical advance opens the possibility to use similar methodologies to isolate and characterize human breast epithelial stem cells, and elucidate their role in regeneration and neoplasia.     

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.     

Stem cells in normal breast development and breast cancer

Abstract.  The main focus of this review is the role of mammary stem cells in normal breast development and carcinogenesis. We have developed a new in vitro culture system that permits, for the first time, the propagation of mammary stem and progenitor cells in an undifferentiated state, which should facilitate the elucidation of pathways that regulate normal mammary stem-cell self-renewal and differentiation. Furthermore, we propose a model in which transformation of stem cells, or early progenitor cells, results in carcinogenesis. A key event in this process is the deregulation of normal self-renewal in these cells. Transformed mammary stem or progenitor cells undergo aberrant differentiation processes that result in generation of the phenotypic heterogeneity found in human and rodent breast cancers. This phenotypic diversity is driven by a small subset of mammary tumour stem cells. We will discuss the important implications of this mammary tumour stem-cell model.     

Isolation and characterization of human mammary stem cells

Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.     

Reprogramming cell fates in the mammary microenvironment

The capacity of any portion of the murine mammary gland to produce a complete functional mammary outgrowth upon transplantation to an epithelium-divested fat pad is unaffected by the age or reproductive history of the donor. Likewise, through serial transplantations, no loss of potency is detected when compared to similar transplantations of the youngest mammary tissue tested. This demonstrates that stem cell activity is maintained intact throughout the lifetime of the animal despite aging and the repeated expansion and depletion of the mammary epithelium through multiple rounds of pregnancy, lactation and involution. These facts support the contention that mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue, to produce dispersed cells results in the desecration of the protection afforded by the “niche” and leads to a reduced capacity of dispersed epithelial cells (in terms of the number transplanted) to recapitulate complete functional mammary structures. Our studies demonstrate 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 cells may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/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.     

Ultrastructure of the putative stem cell niche in rat mammary epithelium

There is now strong evidence that the stem cells of many tissues reside in specialized structures termed niches. The stem cell niche functions to house and regulate symmetric and asymmetric mitosis of stem cells in mammalian skin, mouse and human bone marrow, mouse brain, gut, and hair follicle, and Drosophila ovary and testis. This regulation is effected through the action of various signaling pathways such as Notch, Hedgehog, Wnt and others. The hormones of the estrous cycle, pregnancy and lactation that initiate growth in mouse mammary epithelium appear to act at a paracrine level to regulate mitosis through Notch receptors. Previous work has established that the putative stem cells of the mammary epithelium in several animal species reside near the basement membrane and never make contact with the ductal lumen. We show that these putative stem cells are found in anatomically specialized places created by the cytoplasmic extensions and modifications of neighboring differentiated cells. Such specializations may help to regulate stem cell activity by modulating molecular traffic to putative stem cells and contact with signaling molecules in the basement membrane. The histological characteristics of these putative niches vary as to the kinds of relationships the cells can have with the basement membrane and neighboring cells and as to how many stem or progenitor cells they may contain. This suggests a plasticity that may be relevant to the response of niches to tissue demands, such as wound healing, the periodic growth and regression of mammary epithelium, the process of mammary tumorigenesis therapeutic strategies for breast cancer.     

Regulation of Mammary Stem/Progenitor Cells by PTEN/Akt/β-Catenin Signaling

Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/β-catenin pathway through the phosphorylation of GSK3-β. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/β-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.     

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.     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors, which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype, loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds, which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently, the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore, Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally, we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors.     

Human breast milk is a rich source of multipotent mesenchymal stem cells

Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be “reprogrammed” to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells

Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated in a feeder-independent manner, representing a unique model to study reprogramming and an important step toward establishing a safe, clinical grade of cells for therapeutic use. In this study, we provide a detailed protocol for isolation, preparation and transformation of ASCs from fat tissue into mouse iPSCs in feeder-free conditions and human iPSCs using feeder-dependent or feeder/xenobiotic-free processes. This protocol also describes how ASCs can be used as feeder cells for maintenance of other pluripotent stem cells. ASC derivation is rapid and can be completed in <1 week, with mouse and human iPS reprogramming times averaging 1.5 and 2.5 weeks, respectively.