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.     

Localization of putative stem cells and four cell populations with different differentiation degree in mouse mammary anlagen

The precise localization of putative stem cells and other cells within the embryonic mammary gland would help to elucidate the molecular pathways that govern normal mammary development. The ultrastructural appearances and the antigen of Sca-1 were considered together as putative stem cell markers, and the antigens of cytokeratin, CD10, Muc-1 and CD34 as differentiation markers. Electron microscopy was performed to reveal the ultrastructure of cells in different site of the mammary anlagen. An immunofluorescence system was used to reveal the whole structure profile of the mammary anlagen using the anti-cytokeratin antibody to localize specific types of cell populations such as Sca-1, CD10, Muc-1 and CD34 positive cells within the anlagen, which distribute near the inside edge, distribute uniformly, distribute in the central region and distribute in the site of mesenchyme around the neck of the anlagen, respectively. We also observed under electron microscope that some pale cells like putative stem cells reported by prevenient scholars, which are mainly distributed in the Sca-1⁺ cell population near the inside edge of the anlagen, have pale-stained nucleoplasm and cytoplasm, sparse organelles clustered close to their nucleus and have a lack of rough endoplasmic reticulums and cell polarization. The results indicate that the putative stem cells are localized near the edge of the mammary anlagen; the cell populations with different differentiation degree were localized in the central part and around the edge within the anlagen.     

Identification and characterization of cancer initiating cells from BRCA1 related mammary tumors using markers for normal mammary stem cells

It is hypothesized that cancer stem cells arise either from normal stem cells or from progenitor cells that have gained the ability to self-renew. Here we determine whether mammary cancer stem cells can be isolated by using antibodies that have been used for the isolation of normal mammary stem cells. We show that BRCA1 mutant cancer cell lines contained a subpopulation of CD24+CD29+ or CD24+CD49f+ cells that exhibited increased proliferation and colony forming ability in vitro, and enhanced tumor-forming ability in vivo. The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer. Under low-attachment conditions, we detected “tumorspheres” only in the presence of double positive cells, which maintained their ability to self-renew. Furthermore, CD24+CD29+ cells could form tubular structures reminiscent of the mammary ductal tree when grown in three-dimensional cultures, implying that these cancer cells maintain some of the characteristics of the normal stem cells. Nevertheless, they could still drive tumor formation since as low as 500 double positive cells immediately after sorting from BRCA1 mutant primary tumors were able to form tumors with the same heterogeneity found in the original tumors. These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.     

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.     

Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters

Side population (SP) cells isolated from bone marrow, skeletal muscle, and skin have been shown to engraft in dystrophic muscle. However, there have been questions on the phenotypical heterogeneity, tissue of origin, and relationships among SP cell populations extracted from different tissues. Studies on bone marrow SP cells have followed a consistent protocol for their isolation and results obtained are concordant. In contrast, protocols for the isolation of muscle SP cells vary greatly, and consequently reports on their phenotype, differentiation potential and origin have been inconsistent. To address this controversy, we demonstrate that isolation parameters, such as tissue dissociation, cell counting, Hoechst concentration, and stringency in the selection of SP cells, have an effect on the yield, viability, and homogeneity of SP cells derived from bone marrow, skeletal muscle, and skin. In this paper, we demonstrate that SP cells isolated from the bone marrow are distinct from SP cells extracted from skeletal muscle and skin tissues. This study offers an explanation for the controversy surrounding muscle SP cells, provides a detailed standardized protocol for their isolation, and highlights basic guidelines for reproducible and reliable isolation of SP cells from any tissue.     

Both LRP5 and LRP6 receptors are required to respond to physiological Wnt ligands in mammary epithelial cells and fibroblasts

A canonical Wnt signal maintains adult mammary ductal stem cell activity, and this signal requires the Wnt signaling reception, LRP5. However, previous data from our laboratory have shown that LRP5 and LRP6 are co-expressed in mammary basal cells and that LRP6 is active, leading us to question why LRP6 is insufficient to mediate canonical signaling in the absence of LRP5. Here, we show that at endogenous levels of LRP5 and LRP6 both receptors are required to signal in response to some Wnt ligands both in vitro (in mouse embryonic fibroblasts and mammary epithelial cells) and in vivo (in mammary outgrowths). This subgroup of canonical ligands includes Wnt1, Wnt9b, and Wnt10b; the latter two are expressed in mammary gland. In contrast, the ligand commonly used experimentally, Wnt3a, prefers LRP6 and requires just one receptor regardless of cellular context. When either LRP5 or LRP6 is overexpressed, signaling remains ligand-dependent, but the requirement for both receptors is abrogated (regardless of ligand type). We have documented an LRP5-6 heteromer using immiscible filtration assisted by surface tension (IFAST) immunoprecipitation. Together, our data imply that under physiological conditions some Wnt ligands require both receptors to be present to generate a canonical signal. We have designed a model to explain our results based on the resistance of LRP5-6 heteromers to a selective inhibitor of E1/2-binding Wnt-LRP6 interaction. These data have implications for stem cell biology and for the analysis of the oncogenicity of LRP receptors that are often overexpressed in breast tumors.     

Extracellular proteinase inhibitor-accelerated apoptosis is associated with B cell activating factor in mammary epithelial cells

The expression of extracellular proteinase inhibitor (Expi) gene was induced during the involution of mammary gland, when apoptosis occurs in this tissue. Transient transfection of Expi gene partially induced apoptosis of mammary epithelial HC11 cells. We developed the stable cell lines overexpressing Expi gene and found that overexpression of Expi accelerated apoptosis of mammary epithelial cells under serum starvation. To understand apoptosis pathway involved in the Expi overexpression, we examined the gene expression profile by using apoptosis gene array containing 243 genes. The subsequent confirmation of the altered gene expression by northern analysis demonstrated that overexpression of the Expi gene induced expression of several genes, which included B cell activating factor (BAFF), Bax, cytochrome c, caspase-9, caspase-3, caspase-6, and CIDE-A. From this study, we first demonstrate that BAFF is involved in mammary apoptosis. Furthermore, we have found that the Expi-accelerated apoptosis is mediated via BAFF receptor among three known BAFF receptors: BAFF receptor, tumor necrosis factor (TNF) receptor homologue TACI (transmembrane activator and CAML-interactor), and BCMA (another TNFR homologue, B cell maturation antigen). Our studies also demonstrate that the use of apoptosis array provides an efficient tool to identify apoptosis pathway involved in gene transfection.     

A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule

Bone marrow (BM) from human and rodent species contains a population of multipotential cells referred to as mesenchymal stem cells (MSCs). Currently, MSCs are isolated indirectly by using a culture step and then the generation of fibroblast colony-forming units (CFU-fs). Unprocessed or native BM MSCs have not yet been fully characterised. We have previously developed a direct enrichment method for the isolation of MSCs from human BM by using the CD49a protein (alpha1-integrin subunit). As the CD49a gene is highly conserved in mammals, we have evaluated whether this direct enrichment can be employed for BM cells from rodent strains (rat and mouse). We have also studied the native phenotype by using both immunodetection and immunomagnetic methods and have compared MSCs from mouse, rat and human BM. As is the case for human BM, we have demonstrated that all rodent multipotential CFU-fs are contained within the CD49a-positive cell population. However, in the mouse, the number of CFU-fs is strain-dependent. Interestingly, all rat and mouse Sca-1-positive cells are concentrated within the CD49a-positive fraction and also contain all CFU-fs. In human, the colonies have been detected in the CD49a/CD133 double-positive population. Thus, the CD49a protein is a conserved marker that permits the direct enrichment of BM MSCs from various mammalian species; these cells have been phenotyped as true BM stem cells.     

Nectin-3 expression is elevated in limbal epithelial side population cells with strongly expressed stem cell markers

Corneal epithelial stem cells (CESCs) are essential for maintaining the ocular surface. However, the lack of surface markers for CESCs remains a serious obstacle in the identification of CESCs. Previously, we showed that rabbit limbal epithelial side population (rLE-SP) cells exhibited stem cell phenotypes including increased expression of CD61, a marker for mouse hematopoietic stem cells. Here, we demonstrate that nectin-3, an immunoglobulin-like cell-cell adhesion molecule, is highly expressed in rLE-SP cells. Additionally, nectin-3⁺ cells were significantly enriched among CD61⁺rLE-SP cells as compared to CD61⁻rLE-SP cells. In mouse bone marrow side population cells, a correlation between expression of nectin-3 and CD61 was also observed. These data strongly suggest that nectin-3 may contribute to the identification of CESCs.     

Prominin-1/CD133, a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer

Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (hE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that hE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowmans capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, hE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SPP 1109, CO 298/2-1 to D.C., Hu 275/7-1 to W.B.H.; SPP 1111, Hu 275/8-2 to W.B.H.) and the Fonds der Chemischen Industrie (to W.B.H.)     

Enrichment of corneal epithelial stem/progenitor cells using cell surface markers, integrin alpha6 and CD71

Corneal epithelial stem cells are believed to reside in the basal layer of the limbal epithelium, but no definitive cell surface markers have been identified. For keratinocytes, stem/progenitor cells are known to be enriched by cell surface markers, integrin α₆ and CD71, as a minor subpopulation which shows high integrin α₆ and low CD71 expressions (α₆^bri/CD71^dim). In the present study, we investigated the possibility that corneal epithelial stem cells can be enriched by integrin α₆ and CD71. The α₆^bri/CD71^dim cells were separated by fluorescence-activated cell sorting, as a minor subpopulation of the limbal epithelial cells. They were enriched for relatively small cells, showing a higher clonogenic capacity and expression of stem cell markers, but a lower expression of differentiation markers, compared to other cell populations. The cells were localized immunohistochemically in the basal region of the limbal epithelium. These results indicate that the α₆^bri/CD71^dim subpopulation enriched corneal epithelial stem cells.     

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.     

Lipids noncovalently associated with keratins and other cytoskeletal proteins of mouse mammary epithelial cells in primary culture

Lipids noncovalently associated with cytoskeletal (CS) proteins of mouse mammary epithelial cells (MMEC) grown in primary culture were analyzed. A CS fraction, preapred by subjecting MMEC to 1.5 KCl and 1% Triton X-100 in phosphate buffered saline (pH 7.4), was extracted 4–6 times with chloroform/methanol. Thin-layer chromatography (TLC) indicated that in comparison to whole cell lipid extracts, CS lipids consisted mostly of neutral lipids, especially triacylglycerols and, possibly cholesteryl esters. TLC analysis of chloroform/methanol CS extracts prepared from MMEC that had been incubated 4 h in [³H]palmitate revealed similar results, with the majority of label appearing in triacylglycerols and other neutral lipids. By autoradiography of sodium dodecyl sulfate polyacrylamide gels, all of the major CS proteins appeared labelled. The major regions of autoradiographic density of the gel were excised, the protein solubilized, and the lipids extracted and subjected to TLC. Most of the radiolabel appeared at the origin and ion front and resolved as neutral lipids. In contrast, keratins of 54–55 kDa and 46 kDa appeared to be associated noncovalently with a higher ratio of polar lipids (possibly phospholipids) to nonpolar (neutral lipids). Very little radioactivity, mostly neutral lipid, was associated with actin. A previously unidentified CS component of 30 kDa had primarily noncovalently bound neutral lipid. The results are discussed in terms of the apparent interactions of keratin filaments with the plasma membrane, nuclear envelope and cytoplasmic organelles.     

Prominin-2 is a cholesterol-binding protein associated with apical and basolateral plasmalemmal protrusions in polarized epithelial cells and released into urine

Prominin-2 is a pentaspan membrane glycoprotein structurally related to the cholesterol-binding protein prominin-1, which is expressed in epithelial and non-epithelial cells. Although prominin-1 expression is widespread throughout the organism, the loss of its function solely causes retinal degeneration. The finding that prominin-2 appears to be restricted to epithelial cells, such as those found in kidney tubules, raises the possibility that prominin-2 functionally substitutes prominin-1 in tissues other than the retina and provokes a search for a definition of its morphological and biochemical characteristics. Here, we have investigated, by using MDCK cells as an epithelial cell model, whether prominin-2 shares the biochemical and morphological properties of prominin-1. Interestingly, we have found that, whereas prominin-2 is not restricted to the apical domain like prominin-1 but is distributed in a non-polarized fashion between the apical and basolateral plasma membranes, it retains the main feature of prominin-1, i.e. its selective concentration in plasmalemmal protrusions; prominin-2 is confined to microvilli, cilia and other acetylated tubulin-positive protruding structures. Similar to prominin-1, prominin-2 is partly associated with detergent-resistant membranes in a cholesterol-dependent manner, suggesting its incorporation into membrane microdomains, and binds directly to plasma membrane cholesterol. Finally, prominin-2 is also associated with small membrane particles that are released into the culture media and found in a physiological fluid, i.e. urine. Together, these data show that all the characteristics of prominin-1 are shared by prominin-2, which is in agreement with a possible redundancy in their role as potential organizers of plasma membrane protrusions.     

Lgr5 expression as stem cell marker in human gastric gland and its relatedness with other putative cancer stem cell markers

To explore Lgr5 as the possible stem cell marker in human gastric tissue, 259 normal gastric tissues and dissected gastric adenocarcinoma were analyzed by immunohistochemistry, immunofluorescence double staining and qRT-PCR. The results demonstrated that Lgr5 was expressed in the bottom of the normal gastric gland units, and showed a differential expression in gastric adenocarcinoma with varying differentiation. Lgr5 and Bmi1 were co-expressed within the same cells of gastric glands. CD26 +, CD44 +, ALDH1 + and CD133 + cells co-existed with Lgr5 + cells in the stem cell zone of adjacent normal gastric mucosa, and they were detectable in gastric adenocarcinoma but behaved differently. We concluded that Lgr5 may be the adult stem cell marker in human gastric epithelium; Lgr5 and Bmi1 may belong to the same stem cell population; Lgr5, CD26, CD44, ALDH1, and CD133 may be functionally-associated.     

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G₂/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.     

miR-27a suppresses triglyceride accumulation and affects gene mRNA expression associated with fat metabolism in dairy goat mammary gland epithelial cells

MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides, participate in various biological metabolic processes. miR-27a is a miRNA that is known to regulate fat synthesis and differentiation in preadipocyte cells. However, little is known regarding the role that miR-27a plays in regulating goat milk fat synthesis. In this study, we determined the miR-27a expression profile in goat mammary gland and found that miR-27a expression was correlated with the lactation cycle. Additionally, prolactin promoted miR-27a expression in goat mammary gland epithelial cells. Further functional analysis showed that over-expression of miR-27a down-regulated triglyceride accumulation and decreased the ratio of unsaturated/saturated fatty acid in mammary gland epithelial cells. miR-27a also significantly affected mRNA expression related to milk fat metabolism. Specifically, over-expression of miR-27a reduced gene mRNA expression associated with triglyceride synthesis by suppressing PPARγ protein levels. This study provides the first experimental evidence that miR-27a regulates triglyceride synthesis in goat mammary gland epithelial cells and improves our understanding about the importance of miRNAs in milk fat synthesis.     

Skewed ratios between CD3(+) T cells and monocytes are associated with poor prognosis in patients with HBV-related acute-on-chronic liver failure

Tempering of the innate immune response by T lymphocytes has been demonstrated to play a critical role in protecting animals from inflammation-induced death; however, its role in humans remains unknown. Patients with HBV-related acute-on-chronic liver failure (ACLF) share a striking similarity to the inflammatory response in septic shock where a hyperactive innate response is observed. The present study attempted to characterize the features of CD3⁺ T cells and monocytes and evaluate their clinical implications in 55 patients with HBV-related ACLF, 30 patients with chronic hepatitis B (CHB) and 30 healthy controls (HC). We found that the ratio between circulating CD3⁺ T cells and monocytes (T/M) was decreased in ACLF patients, due to decreased CD3 counts and increased monocyte counts compared with CHB and HC subjects. We also found that the T/M ratios were decreased from the early to the intermediate stage and reached the lowest value at the late stage in ACLF patients. Analyses with clinical parameters revealed that T/M ratios were negatively correlated with the Model for End-Stage Liver Disease Score and direct bilirubin, and positively correlated with prothrombin activity. Moreover, increased T/M ratios were observed in patients with good prognosis, but not in patients with a poor outcome; and ACLF patients who received liver transplantation exhibited an increased T/M ratio. Importantly, we found that programmed death-1 receptor (PD-1) was drastically upregulated on both CD4⁺ T and CD8⁺ T cells in ACLF, which at least in part contributed to the T-cell loss in these patients. Mechanically, the in vitro co-culture assay revealed that both CD4⁺ T and CD8⁺ T cells, as well as regulatory T cells, could inhibit TNF-α secretion by monocytes. In addition, the TNF-α levels in ACLF serum were negatively correlated with T/M ratios. In conclusion, our study identified the novel potential role of T/M ratio in predicting disease progression and provided novel evidences for further studies of the immunopathogenesis in ACLF.