Single-cell RNA sequencing of freshly isolated bovine milk cells and cultured primary mammary epithelial cells

AbstractBovine mammary function at molecular level is often studied using mammary tissue or primary bovine mammary epithelial cells (pbMECs). However, bulk tissue and primary cells are heterogeneous with respect to cell populations, adding further transcriptional variation in addition to genetic background. Thus, understanding of the variation in gene expression profiles of cell populations and their effect on function are limited. To investigate the mononuclear cell composition in bovine milk, we analyzed a single-cell suspension from a milk sample. Additionally, we harvested cultured pbMECs to characterize gene expression in a homogeneous cell population. Using the Drop-seq technology, we generated single-cell RNA datasets of somatic milk cells and pbMECs. The final datasets after quality control filtering contained 7,119 and 10,549 cells, respectively. The pbMECs formed 14 indefinite clusters displaying intrapopulation heterogeneity, whereas the milk cells formed 14 more distinct clusters. Our datasets constitute a molecular cell atlas that provides a basis for future studies of milk cell composition and gene expression, and could serve as reference datasets for milk cell analysis.

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Single cell transcriptome atlas of mouse mammary epithelial cells across development

Breast Cancer Research ◽

10.1186/s13058-021-01445-4 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Bhupinder Pal ◽

Yunshun Chen ◽

Michael J. G. Milevskiy ◽

François Vaillant ◽

Lexie Prokopuk ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Developmental Stages ◽

Mammary Epithelial Cells ◽

Expression Profiles ◽

Single Cells ◽

Chromatin Accessibility ◽

Mammary Epithelial ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

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Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland

Journal of Cell Science ◽

10.1242/jcs.112.11.1771 ◽

1999 ◽

Vol 112 (11) ◽

pp. 1771-1783 ◽

Cited By ~ 6

Author(s):

A.D. Metcalfe ◽

A. Gilmore ◽

T. Klinowska ◽

J. Oliver ◽

A.J. Valentijn ◽

...

Keyword(s):

Cell Death ◽

Mammary Gland ◽

Epithelial Cells ◽

De Novo ◽

Mammary Epithelial Cells ◽

Expression Profiles ◽

Mammary Epithelial ◽

Mammary Tissue ◽

Pregnancy And Lactation

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

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Examination of methionine stimulation of gene expression in dairy cow mammary epithelial cells using RNA-sequencing

Journal of Dairy Research ◽

10.1017/s0022029920000199 ◽

2020 ◽

Vol 87 (2) ◽

pp. 226-231

Author(s):

Xiaoming Hou ◽

Minghui Jiang ◽

Jinyu Zhou ◽

Shuyuan Song ◽

Feng Zhao ◽

...

Keyword(s):

Gene Expression ◽

Protein Synthesis ◽

Epithelial Cells ◽

Rna Sequencing ◽

Milk Protein ◽

Mammary Epithelial Cells ◽

Cell Model ◽

Expression Profiles ◽

Mammary Epithelial ◽

Bovine Mammary Epithelial Cells

AbstractIn this research communication, a cell model with elevated β-CASEIN synthesis was established by stimulating bovine mammary epithelial cells with 0.6 mM methionine, and the genome-wide gene expression profiles of methionine-stimulated cells and untreated cells were investigated by RNA sequencing. A total of 458 differentially expressed genes (DEGs; 219 upregulated and 239 downregulated) were identified between the two groups. Gene Ontology (GO) analysis showed that the two highest-ranked GO terms in ‘molecular function’ category were ‘binding’ and ‘catalytic activity’, suggesting that milk protein synthesis in methionine-stimulated cells requires induction of gene expression to increase metabolic activity. Kyoto Encyclopedia of Genes and Genomes analysis revealed that within the ‘environmental information processing’ category, the subcategory that is most highly enriched for DEGs was ‘signal transduction’. cGMP-PKG, Rap1, calcium, cAMP, PI3K-AKT, MAPK, and JAK-STAT are the pathways with the highest number of DEGs, suggesting that these signaling pathways have potential roles in mediating methionine-induced milk protein synthesis in bovine mammary epithelial cells. This study provides valuable insights into the physiological and metabolic adaptations in cells stimulated with methionine. Understanding the regulation of this transition is essential for effective intervention in the lactation process.

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Host–pathogen gene expression profiles during infection of primary bovine mammary epithelial cells with Escherichia coli strains associated with acute or persistent bovine mastitis

Veterinary Microbiology ◽

10.1016/j.vetmic.2011.08.016 ◽

2012 ◽

Vol 155 (2-4) ◽

pp. 291-297 ◽

Cited By ~ 13

Author(s):

O. Kerro Dego ◽

S.P. Oliver ◽

R.A. Almeida

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Expression Profiles ◽

Bovine Mastitis ◽

Gene Expression Profiles ◽

Mammary Epithelial ◽

Bovine Mammary Epithelial Cells ◽

Host Pathogen

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Broad-Spectrum Activity against Bacterial Mastitis Pathogens and Activation of Mammary Epithelial Cells Support a Protective Role of Neutrophil Cathelicidins in Bovine Mastitis

Infection and Immunity ◽

10.1128/iai.01090-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1781-1788 ◽

Cited By ~ 47

Author(s):

Linda Tomasinsig ◽

Gennyfer De Conti ◽

Barbara Skerlavaj ◽

Renata Piccinini ◽

Maria Mazzilli ◽

...

Keyword(s):

Epithelial Cells ◽

Broad Spectrum ◽

Mammary Epithelial Cells ◽

Bovine Milk ◽

Bovine Mastitis ◽

Defense Responses ◽

Mammary Epithelial ◽

Host Cells ◽

Mammary Tissue ◽

Necrosis Factor Alpha

ABSTRACT Cathelicidins are peptide components of the innate immune system of mammals. Apart from exerting a direct antibiotic activity, they can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied, but there is a lack of published information on their expression and activities in the context of mastitis. The aims of this study were to investigate the expression of the bovine cathelicidins BMAP-27, BMAP-28, Bac5, and indolicidin in healthy and infected mammary tissue and in lipopolysaccharide (LPS)-treated cells, to determine their activities against bacteria isolated from bovine mastitis, and to examine their potentials to trigger defense responses in bovine mammary cells. The genes were found to be upregulated in LPS-stimulated neutrophils, but not in infected quarters or epithelial cells. All peptides showed a variably broad spectrum of activity against 28 bacterial isolates from bovine mastitis (MIC values, 0.5 to 32 μM), some of which were antibiotic resistant. The activity of each peptide was significantly enhanced when it was pairwise tested with the other peptides, reaching the synergy threshold when indolicidin was present. The bactericidal activity was sensitive to milk components; BMAP-27 and -28 were highly effective in mastitic bovine milk and inhibited in milk from healthy cows. Both peptides were also active in whey and in blood serum and triggered the expression of tumor necrosis factor alpha (TNF-α) in bovine mammary epithelial cells. Our results indicate multiple roles for the bovine cathelicidins in mastitis, with complementary and mutually enhanced antimicrobial activities against causative pathogens and the capacity to activate host cells.

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Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland

Journal of Mammary Gland Biology and Neoplasia ◽

10.1007/s10911-021-09486-3 ◽

2021 ◽

Author(s):

Samantha Henry ◽

Marygrace C. Trousdell ◽

Samantha L. Cyrill ◽

Yixin Zhao ◽

Mary. J. Feigman ◽

...

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Epithelial Cells ◽

Single Cell ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Cellular Heterogeneity ◽

Molecular Signatures ◽

Mammary Cells ◽

Human Breast

AbstractThe developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.

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A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

Molecular Endocrinology ◽

10.1210/me.2008-0097 ◽

2008 ◽

Vol 22 (12) ◽

pp. 2677-2688 ◽

Cited By ~ 45

Author(s):

Paul G. Tiffen ◽

Nader Omidvar ◽

Nuria Marquez-Almuina ◽

Dawn Croston ◽

Christine J. Watson ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Oncostatin M ◽

Specific Gene ◽

Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

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Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.84.1.136 ◽

1987 ◽

Vol 84 (1) ◽

pp. 136-140 ◽

Cited By ~ 312

Author(s):

M. L. Li ◽

J. Aggeler ◽

D. A. Farson ◽

C. Hatier ◽

J. Hassell ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Basement Membrane ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Casein Gene

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Bovine mammary epithelial cells, initiators of innate immune responses to mastitis

Australian Journal of Experimental Agriculture ◽

10.1071/ea05046 ◽

2005 ◽

Vol 45 (8) ◽

pp. 757 ◽

Cited By ~ 15

Author(s):

C. Gray ◽

Y. Strandberg ◽

L. Donaldson ◽

R. L. Tellam

Keyword(s):

Immune Response ◽

Mammary Gland ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Vital Role ◽

Mammary Epithelial ◽

Innate Immune ◽

Mammary Tissue ◽

Effector Cells ◽

Bovine Mammary Epithelial Cells

Innate immunity plays a vital role in the protection of the bovine mammary gland against mastitis. Until recently, the migration of effector cells such as neutrophils and monocytes into the mammary gland was thought to provide the only defence against invading pathogens. However, mammary epithelial cells may also play an important role in the immune response, contributing to the innate defence of the mammary tissue through secretion of antimicrobial peptides and attraction of circulating immune effector cells. This paper reviews the innate immune pathways in mammary epithelial cells and examines their role in the initiation of an innate immune response to Gram-positive and Gram-negative bacteria.

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