Comparison of FACS-isolated mammary epithelial populations (CD49f/CD61)
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ABSTRACT:
PROVIDER: PRJNA853576 | ENA |
REPOSITORIES: ENA
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Project description:To validate the CD49f/CD61 flow cytometry panel for resolving the three major mammary epithelial lineages, mammary epithelial cells from MMTV-Cre mice were collected by FACS and analyzed by RNA-seq for comparison to previously published single-cell RNA-seq studies, which non-biasedly identified the three major lineages: basal, alveolar progenitor (AP), and hormone-sensing luminal (HSL) cells.
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Project description:To delineate epithelial subpopulations in human mammary tissue, hematopoietic and endothelial cells were depleted from freshly isolated cell suspensions derived from reduction mammoplasties by fluorescence-activated cell sorting. The resultant Lin- population was fractionated into four distinct subpopulations using CD49f (α6-integrin) and epithelial cell adhesion molecule (EpCAM; also referred to as CD326 and ESA). Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as fibroblast-enriched stromal (CD49f -EpCAM-), mammary stem cell (MaSC)-enriched (CD49f hiEpCAM-), luminal progenitor (CD49f +EpCAM+), and mature luminal (CD49f –EpCAM+) cell subpopulations. Microarray profiling was used to derive gene expression signatures representative of these subpopulations using freshly sorted cells (>90% purity) from normal breast tissue. The four mammary cell subpopulations were found to have distinct gene expression profiles.
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Project description:To delineate epithelial subpopulations in human mammary tissue, hematopoietic and endothelial cells were depleted from freshly isolated cell suspensions derived from reduction mammoplasties by fluorescence-activated cell sorting. The resultant Lin- population was fractionated into four distinct subpopulations using CD49f (α6-integrin) and epithelial cell adhesion molecule (EpCAM; also referred to as CD326 and ESA). Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as fibroblast-enriched stromal (CD49f -EpCAM-), mammary stem cell (MaSC)-enriched (CD49f hiEpCAM-), luminal progenitor (CD49f +EpCAM+), and mature luminal (CD49f âEpCAM+) cell subpopulations. Microarray profiling was used to derive gene expression signatures representative of these subpopulations using freshly sorted cells (>90% purity) from normal breast tissue. The four mammary cell subpopulations were found to have distinct gene expression profiles. Four mammary cell subpopulations from three individual patient samples were analyzed.
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Project description:A FACS protocol is described that eliminates isolation and staining artifacts to allow accurate comparison between cell populations isolated from organs obtained from disparate mouse groups. This protocol was validated by characterizing the estrogen receptor positive cells within the mammary gland of transgenic mice with different genotypes at different stages of the estrous cycle. We include protocols necessary to batch stage animals within the cycle to proceed directly to FACS, which provides optimal RNA yields for RNA-seq. For complete details on the use and execution of this protocol, please refer to Ludwik et al. (2020).
| S-EPMC7811174 | biostudies-literature