Dataset Information

Quantification of regenerative potential in primary human mammary epithelial cells

ABSTRACT: We present an organoid regeneration assay in which freshly dissociated human mammary epithelial cells from healthy donors are grown in adherent/rigid or floating/compliant collagen I gels. In both conditions, luminal progenitors (CD49f+EpCAM+) form spheres, whereas basal cells (CD49fhiEpCAM-) generate branched ductal structures. However, in compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Moreover, the membrane metallo-endopeptidase CD10 as an additional surface marker enriches for TDLU-formation and reveals the presence of stromal cells within the CD49fhiEpCAM- population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. Comparison of three cell populations derived from FACS-sorted primary human mammary epithelial cells: luminal progenitors, CD10+ basal cells and CD10- basal cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Steffen Sass Jelena R Linnemann Christina H Scheel Martin Irmler Lisa K Meixner Uwe J Kloos Benjamin Hirschi Christian Gabka Harald S Bartsch Johannes Beckers Haruko Miura Karl Sotlar Fabian Theis

PROVIDER: E-GEOD-64248 | ArrayExpress | 2015-07-02

SECONDARY ACCESSION(S): GSE64248PRJNA270533

REPOSITORIES: GEO, ArrayExpress

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Publications

Quantification of regenerative potential in primary human mammary epithelial cells.

Linnemann Jelena R JR Miura Haruko H Meixner Lisa K LK Irmler Martin M Kloos Uwe J UJ Hirschi Benjamin B Bartsch Harald S HS Sass Steffen S Beckers Johannes J Theis Fabian J FJ Gabka Christian C Sotlar Karl K Scheel Christina H CH

Development (Cambridge, England) 20150612 18

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is re ...[more]

PMID: 26071498

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Project description:Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. The goals and objectives of this study were 1.) To generate gene expression profiles and identify signatures of freshly isolated, non-cultured, luminal epithelial or basal epithelial and CD10 positive or negative stromal cell populations from the human breast. 2.) Use the generated signatures to characterize and validate the molecular identity of human primary epithelial and stromal/mesenchymal breast cells maintained long-term in novel ex vivo culture conditions in serum free medium (previous study in our lab) Breast tissue from three different reduction mammoplasties was dissociated and single cells were labeled with antibodies against lineage markers (CD31, CD45 CD235ab), CD10, EpCAM, CD49f and DAPI. Different cell populations were identified, separated and sorted with a fluorescence activated cell sorter. Gene expression profiling of luminal epithelial (EpCAM+, CD49f+/-), basal epithelial (EPCAM-, CD49f+), CD10 negative and CD10 positive stromal cells was performed and distinct gene expression signatures for the four cell populations were identified. Overall design: A total of 12 samples were analyzed. To obtain three complete biological replicates, breast tissue from three different reduction mammoplasties (MP9, MP13, MP15) was dissociated and single cells were labeled with fluorescent antibodies prior to cell sorting. Four distinct cell populations (two epithelial and two stromal) were identified and gated. 300 cells of each of the four cell populations were sorted directly in 100ul TRIZOL for all three donor tissues. The samples were immediately frozen on dry ice and stored at -70°C prior to RNA extraction. Gene expression profiles of luminal epithelial cells (EpCAM+, CD49f+/-), basal epithelial (EPCAM-, CD49f+), CD10 negative and CD10 positive stromal cells were successfully obtained for all three reduction mammoplasties.