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.
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.
Project description:During gestation, alveolar cells are derived from luminal progenitors in the mammary gland. However, the mechanism underlying luminal progenitor commitment to alveolar cells remains largely unknown. By using five genetically modified mouse lines and single cell RNA sequencing, we identified a Kindlin-2 - Stat3 - Dll1 signaling cascade in myoepithelial cells which controls the inactivation of Notch signaling in luminal cells that consequently drives luminal progenitor commitment to alveolar cells. We found that loss of Kindlin-2 in myoepithelial cells impairs mammary morphogenesis and alveologenesis, and lactation. Single-cell profiling reveals that Kindlin-2 loss significantly decreases the proportion of alveolar cells.
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.
Project description:Highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue were isolated and compared their transcriptomes which were obtained using PCR-Long-SAGE technology. Keywords: mammary progenitors, stem cells, Notch signaling, gene expression Four SAGE libraries were constructed on RNA samples extracted from highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells isolate from a normal human mammary tissue.
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.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.