Single-cell RNA sequencing of mammary epithelial and myoepithelial cells
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ABSTRACT:
PROVIDER: PRJNA1173731 | ENA |
REPOSITORIES: ENA
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Project description:Differentiation of stem cells embedded within the mammary epithelium is orchestrated by lineage-specifying transcription factors. Unlike the well-defined luminal hierarchy, dissection of the basal lineage has been hindered by a lack of specific markers. Inhibitor of Differentiation 4 (ID4) is a basally-restricted helix-loop-helix (HLH) transcription factor essential for mammary development. Here we show that ID4 is highly expressed in basal stem cells and decreases during myoepithelial differentiation. By integrating transcriptomic, proteomic and chromatin-association data we reveal that ID4 is required to suppress myoepithelial gene expression and cell fate.
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Project description: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. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in DFCI-1 medium retain a fraction with progenitor cell properties. These cells co-express basal, luminal and stem/progenitor cell markers. Clonal derivatives of progenitors co-expressing these markers fall into two distinct types: K5+/K19- (Type I) and K5+/K19+ (Type II). We show that both types of progenitor cells have self-renewal and differentiation ability. Through microarray analysis, we want to identify genes and pathways linked to human mammary epithelial stem/progenitor cell self-renewal and differentiation. Normal human mammary epithelial cells (hMECs) were isolated from Reduction Mammoplasty and immortalized by hTERT. Type I K5+/K19- and Type II K5+/K19+ cell colonies were isolated from hTERT-immortalized hMECs and cultured in MEGM medium for self-renewal and differentiation. Total RNA isolated from Type I, Type II, and differentiated Myoepithelial (Myo) cells were used on Affymetrix microarrays.
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Project description:Single Cell RNA Sequencing of Mouse Mammary Epithelial Cells
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Project description:Single Cell RNA-sequencing of mammary gland epithelial cells
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Project description:A comparison of the mRNAs analysis in lactation mammary myoepithelial cells (GFP+/GFP-) and brown adipocytes (GFP+) from Myf5-Cre-ROSAmTmG and Ucp1-iCre-ROSAmTmG mice. Results provide the gene expression signature in the brown origin (UCP1/Myf5-positive) myoepithelial cells in vivo.
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Project description:The basal cell lineage in the mouse mammary gland is perceived to be composed mostly of differentiated myoepithelial cells intermixed with a rare subpopulation of mammary stem cells. Here, we show that a high proportion (~30%) of basal cells have colony forming capacity when actin-myosin contraction is prevented. A basal cell subpopulation enriched for mammary repopulating units (MRUs) is shown to be enriched for dividing cells. Inducing basal cells to proliferate in vitro increases MRU frequency 10-fold and permits a ~750-fold expansion of MRUs. Transplantation of single-cell-derived basal colonies into cleared fat pads demonstrates that the majority (~80%) of these colonies can repopulate a mammary gland. This suggests that a high proportion of basal cells, most of which were previously perceived to be terminally differentiated myoepithelial cells, have latent mammary stem cell potential. Mammary glands from 10-14 week old, female, virgin C57BL/6J mice were dissociated into single cells and basal cells were sorted by flow cytometry at a purity of ~95%. Inducing basal cell proliferation in vitro causes mammary stem cell expansion over 7 days in culture. Basal cells were cultured on Matrigel-coated cell culture plastic in a keratinocyte stem cell media supplemented with the Rho kinase inhibitor Y-27632 and were co-seeded with irradiated NIH 3T3 fibroblast feeder cells. In order to determine the molecular changes that occur during culture that may contribute to mammary stem cell expansion, gene expression profiling was conducted on basal cells pre-culture and after 1- or 7-day-culture.
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Project description:To get molecular insight into age- and compartment-specific changes in telomere maintenance and associated properties in human mammary gland, we analyzed distinct subsets of normal human mammary epithelial cells. The cells were isolated by fluorescent activated cell sorting (FACS) directly from mammary tissue obtained from normal women undergoing reduction mammoplasties with concomitant removal of hematopoietic and endothelial cells by depletion of CD45pos and CD31pos cells. The three epithelial cell populations then isolated were: (i) CD49fhiEPCAMneg/low cells, (ii) CD49fposEPCAMpos cells and (iii) CD49fnegEPCAMpos cells. The CD49fhiEPCAM-/low cells are selectively enriched in mammary stem cells with functional mammary gland regenerating activity in suitably transplanted immunodeficient mice, bipotent progenitors that form colonies of adherent myoepithelial and luminal cells in vitro, myoepithelial-restricted progenitors that form colonies of exclusively adherent myoepithelial cells in vitro, and mature myoepithelial cells that are not clonogenic (collectively referred to as basal cells, BCs). The CD49fposEPCAMpos cells are selectively enriched in luminal progenitors (referred to as luminal progenitors, LPs); and the CD49fnegEPCAMpos cells are selectively enriched in mature luminal cells (referred to as luminal cells, LCs). Differences in gene expression in general and telomere associated genes in particular were elucidated by analyzing mammary epithelial subpopulations. Total RNA was isolated from 24 samples obtained from FACS purification of mammary epithelial subpopulations from 9 reduction mammoplasty breast tissues. Global gene expression profiling was performed by array.
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