1,721,033 research outputs found
Functional identification of bovine mammary epithelial stem/progenitor cells
No full textThe in vitro and in vivo assays have been developed to detect primitive bovine mammary cells.
The in vitro CFC assay allowed us to detect different type of progenitors as demonstrated by the
different colonies that they were able to generate when cultured in vitro.
Cells from bovine mammary tissue were able to generate organized outgrowths in a xenograft model.
Moreover progenitor cells could be detected in these outgrowths, but the frequencies of the different
type of colonies showed a marked variation when compared to the CFC assay performed on freshly
dissociated BMECs.
The systems described here provide very useful tools to improve the knowledge of the bovine
mammary tissue hierarchy and of the mechanisms and factors that drive proliferation and
differentiation. As future perspective this knowledge may then be used to develop new strategies for
increasing milk production in dairy cows by specifically targeting signaling pathways that are important
in the control of the stem/progenitor cell compartmen
Bovine mammary stem cells: new perspective for dairy science
No full textMammary stem cells provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of mammary stem cells can potentially benefit milk yield, persistency, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and alter the function of bovine mammary stem cells. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. Interestingly, different studies reported recently the identification of putative mammary stem cells in human breast milk and new data are available in ruminants for this issue. In this review we summarized the main achievements in this field for dairy cow science and describe the interesting perspectives open to manipulate milk persistency during lactation and to cope with oxidative stress during the transition period. The exciting possibility that stem cell expansion can influence milk production is currently under investigation. The identification of primitive cell types within cow’s milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications
Epidermal growth factor receptor and hepatocyte growth factor receptor act as redundant but independent inputs to promote survival to mammary epithelial cells and breast cancer cells
No full textBreast cancer is the most common cancer and the second leading cause of cancer mortality in women with approximately one in eight being affected in their lifetime. Breast cancer is a complex and heterogeneous disease, with at least, five currently described subtypes that show different clinical course and response to therapeutic agents. Targeted therapies that inhibit receptor tyrosine kinases (RTKs) and the downstream signaling pathways have shown promising anticancer activity, but their efficacy in many solid tumors, including breast cancer, has been modest possibly because coactivation of different RTKs affects the response of tumor cells to targeted therapies.
In this work we have analyzed the dependence of mammary epithelial cells and mammary tumor derived cell lines to RTK inhibition and how the downstream signaling is modulated. We found that MCF-10A and NMuMG as well as ERBB2 tumor derived cell lines are addicted to Epidermal Growth Factor Receptor (EGFR) for proliferation and survival. EGFR inhibition completely suppressed the ERK1/2 and the AKT pathways in these cell lines. Hepatocyte Growth Factor receptor (MET), but not other RTKs like insulin-like growth factor receptor or fibroblast growth factor receptor, was the only RTK that rendered anti-EGFR inhibition ineffective in extinguishing downstream signaling by replacing activated EGFR in the PI3K and MAPK complexes and by restoring cell viability and proliferation
Identification of Goat Mammary Stem/Progenitor Cells
No full textGoat mammary gland epithelial cells have been used to establish primary and permanent cell lines, but to date, no data have been available regarding mammary stem cells (MaSCs) in this species. The detection and characterization of goat MaSCs is an important task for a better understanding of the cyclic character of mammary gland development, which will also offer the potential for manipulation of lactation yield and persistency. The objective of the present study was to demonstrate that a subpopulation of goat MaSCs resides in the goat mammary gland. Mammary tissue from lactating Saanen goats (Capra hircus) was dissociated and processed to a single-cell suspension. Using an in vitro colony-forming assay, we demonstrated that distinct colony types, which expressed specific lineage markers, arose from unipotent progenitors. Using two different growth media, we showed that the frequencies of caprine clonogenic progenitors differed according to growth conditions. Goat epithelial cells were transplanted under the kidney capsule of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, where they formed organized, bilayered structures. Our results indicate the presence of goat MaSCs in the caprine mammary gland. To our knowledge, these data represent the first description of the tissue hierarchy of the goat mammary gland and demonstrate the regenerative potential of adult goat MaSCs
Human milk protein production in xenografts of genetically engineered bovine mammary epithelial stem cells
No full textBACKGROUND:
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
Mechanisms of modulation of the Egr gene family in mammary epithelial cells of different species
Full text linkIn the adult female, within the estrous cycle, the mammary gland undergoes multiple rounds of growth, with increased cellular proliferation, and involution, with increased apoptosis. The increase in proliferation is elicited by endocrine (Estrogen, Progesterone), as well as locally produced (epidermal growth factor, insulin-like growth factor, etc) growth factors. Among the genes that are modulated during cellular proliferation, immediate early genes play a fundamental role, being rapidly upregulated and then downregulated within the G0/G1 phase of the cell cycle, allowing the progression to the subsequent phases. Egrs (1-4) are immediate early genes that encode for transcription factors that promote, within different cell types and depending on the strength and duration of the stimuli, several different responses like mitogenesis, differentiation, apoptosis or even anti-apoptosis. In this work we have studied the mechanisms of modulation of the Egr family, in mammary epithelial cells of different origin (bovine, canine, feline, murine). Following stimulation with growth medium, Egr mRNA expression showed a strong upregulation reaching a peak at 45-60min, that rapidly declined. Among several cytokines, particularly important for mammary morphogenesis, that we have tested (EGF, IGF-I, insulin, estrogen, progesterone), only EGF upregulated Egrs to levels close to those elicited by growth medium. In order to understand how the Egr transcription factors were regulated, we have inhibited Erk 1/2 and PI3K, molecules that drive two major intracellular signaling pathways. Inhibition of the Erk 1/2 pathway totally abolished Egr upregulation mediated by growth medium or EGF. On the other hand, the PI3K-Akt pathway played a minor role on Egr levels, with a strong inhibitory effect on cat GH2 cells only, that could be ascribed to reduced Erk phosphorylation following PI3K inhibition. Finally we showed that addition of growth medium also upregulated that the mammary luminal marker cytokeratin 18, but only in the murine NMuMG cell line. This is the first manuscript describing how the Egr transcription factors are expressed in mammary epithelial cells of domestic animals and which growth factors and signaling pathways modulate their expression
MODULATION OF IMMEDIATE EARLY GENES IN MAMMARY EPITHELIAL CELLS
No full textIn the mammary gland, during the different phases of the reproductive cycle, moments of tissue growth
with increased cellular proliferation are followed by moments of tissue involution, with augmented cellular apoptosis. These phases are regulated both by endocrine hormones (estrogens, progesterone, etc) and by local acting growth factors (EGFs, IGFs, etc). The downstream cellular signaling of these factors induces the expression of immediate early genes (IEGs) and late genes that monitor different steps within the cell cycle. An altered cellular response to these signals may cause deregulated proliferation within this tissue, with increased cancer susceptibility. The aim of our work is to understand, in mammary epithelial cells, the expression kinetics of three
important IEGs (EGR1, FOS, JUN) in response to different stimuli. We are also focusing our research on which intracellular signaling pathway, involved in mammary growth or differentiation, is able to modulate the expression of EGR1, FOS and JUN. Mammary epithelial cells obtained from different species of domestic animals are grown in a starving
medium in order to obtain a synchronization in the G0/G1 phase of the cell cycle. Subsequently cells are stimulated with various stimuli (growth medium, estrogen, progesterone, EGF, IGFs, etc.) at different time points and IEGs expression is analyzed by real-time PCR and/or western-blot. Treatment with UO126 (ERK 1/2 inhibitor) and/or Wortmannin (PI3K-akt inhibitor) in combination with different stimuli are used to investigate the relative importance of these two important signaling pathways on IEGs RNA and protein expression. We have observed that expression of EGR1 and FOS is strongly upregulated following stimulation with
growth medium, EGF and phorbol 12-myristate 13-acetate. JUN is also upregulated, but to a lower levels. Following addition of growth medium we have observed an increase in EGR1 protein expression associated with ERK1/2 and PI3K-akt signaling pathway activation. When ERK1/2 inhibitor is added together to stimuli that upregulate IEGs we observe that UO126 abolishes EGR1 and FOS upregulation leaving JUN expression mostly unaltered. Western blot analysis suggests a similar trend in EGR1 protein expression. This result indicates that IEGs upregulation following phorbol 12-myristate 13-acetate (an activator of protein kinase C which is downstream G-protein coupled receptors) stimulus is driven exclusively by ERK1/2 phosphorilation. PI3K-Akt pathway inhibition with Wortmannin does not influence IEGs RNA expression and EGR1 protein levels. We conclude that the ERK1/2, but not the PI3K-akt pathway, is the most important signaling event that drives the modulation of both EGR1 and FOS in mammary epithelial cells of domestic animals. On the other hand, JUN Expression is probably controlled through different signaling pathways
Clonogenic assay allows for selection of a primitive mammary epithelial cell population in bovine
Full text linkAdult mammary stem cells have been identified in several species including the bovine. They are responsible for the development of the gland and for cyclic remodeling during estrous cycles and pregnancy. Epithelial cell subpopulations exist within the mammary gland. We and others showed previously that the Colony Forming Cell (CFC) assay can be used to detect lineage-restricted mammary progenitors. We carried out CFCs with bovine mammary cells and manually separated colonies with specific morphologies associated with either a luminal or a myoepithelial phenotype. Expression of specific markers was assessed by immunocytochemistry or by flow cytometry to confirm that the manual separation resulted in isolation of phenotipically different cells. When transplanted in recipient immunodeficient mice, we found that only myoepithelial-like colonies gave rise to outgrowths that resembled bovine mammary alveoli, thus proving that adult stem cells were maintained during culture and segregated with myoepithelial cells. After recovery of the cells from the transplanted mice and subsequent progenitor content analysis, we found a tendency to detect a higher progenitor frequency when myoepithelial-like colonies were transplanted. We here demonstrate that bovine adult mammary stem cells can be sustained in short-term culture and that they can be enriched by manually selecting for basal-like morphology
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