Quantification of regenerative potential in primary human mammary epithelial cells

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 required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− 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. Summary: An assay in which single, freshly isolated human mammary epithelial cells are cultured in a matrix environment is developed and used to quantify the regenerative potential of human mammary cells.

[1]  Li Yang,et al.  Identification of multipotent mammary stem cells by protein C receptor expression , 2014, Nature.

[2]  H. Kleinman,et al.  Matrigel: from discovery and ECM mimicry to assays and models for cancer research. , 2014, Advanced drug delivery reviews.

[3]  David J Mooney,et al.  Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. , 2014, Nature materials.

[4]  S. Menon,et al.  Mammary stem cells have myoepithelial cell properties , 2014, Nature Cell Biology.

[5]  J. Visvader,et al.  Mammary stem cells and the differentiation hierarchy: current status and perspectives , 2014, Genes & development.

[6]  Bogi Andersen,et al.  Mammary morphogenesis and regeneration require the inhibition of EMT at terminal end buds by Ovol2 transcriptional repressor. , 2014, Developmental cell.

[7]  I. Martin,et al.  TGF-β-induced differentiation into myofibroblasts involves specific regulation of two MKL1 isoforms , 2014, Development.

[8]  Jane E. Visvader,et al.  In situ identification of bipotent stem cells in the mammary gland , 2014, Nature.

[9]  Sandro Santagata,et al.  Taxonomy of breast cancer based on normal cell phenotype predicts outcome. , 2014, The Journal of clinical investigation.

[10]  Andrew J. Ewald,et al.  Collective Invasion in Breast Cancer Requires a Conserved Basal Epithelial Program , 2013, Cell.

[11]  D. Geelen,et al.  In Vitro Propagation , 2013 .

[12]  V. Weaver,et al.  Strength Under Tension , 2013, Science.

[13]  C. Eaves,et al.  Developmental Changes in the in Vitro Activated Regenerative Activity of Primitive Mammary Epithelial Cells , 2013, PLoS biology.

[14]  Wassim Raffoul,et al.  Progesterone/RANKL Is a Major Regulatory Axis in the Human Breast , 2013, Science Translational Medicine.

[15]  J. Visvader,et al.  Remodeling of the lymphatic vasculature during mouse mammary gland morphogenesis is mediated via epithelial-derived lymphangiogenic stimuli. , 2012, The American journal of pathology.

[16]  C. Caldas,et al.  Phenotypic and functional characterisation of the luminal cell hierarchy of the mammary gland , 2012, Breast Cancer Research.

[17]  M. Detmar,et al.  Interaction of tumor cells and lymphatic vessels in cancer progression , 2012, Oncogene.

[18]  R. Nusse,et al.  Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. , 2012, Cell stem cell.

[19]  J. Debnath,et al.  Cyclic AMP regulates formation of mammary epithelial acini in vitro , 2012, Molecular biology of the cell.

[20]  C. Brisken,et al.  ER and PR signaling nodes during mammary gland development , 2012, Breast Cancer Research.

[21]  Wenjun Guo,et al.  Slug and Sox9 Cooperatively Determine the Mammary Stem Cell State , 2012, Cell.

[22]  J. Rosen,et al.  On hormone action in the mammary gland. , 2012, Cold Spring Harbor perspectives in biology.

[23]  A. Weiss,et al.  Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival , 2011, Proceedings of the National Academy of Sciences.

[24]  O. Petersen,et al.  Mammary gland stem cells: current status and future challenges. , 2011, The International journal of developmental biology.

[25]  A. Rocha,et al.  Distinct stem cells contribute to mammary gland development and maintenance , 2011, Nature.

[26]  Patricia J Keely,et al.  Mammary gland ECM remodeling, stiffness, and mechanosignaling in normal development and tumor progression. , 2011, Cold Spring Harbor perspectives in biology.

[27]  S. Schnitt,et al.  Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines , 2010, Breast Cancer Research.

[28]  J. Muschler,et al.  Cell-matrix interactions in mammary gland development and breast cancer. , 2010, Cold Spring Harbor perspectives in biology.

[29]  C. Caron de Fromentel,et al.  The CD10 Enzyme Is a Key Player to Identify and Regulate Human Mammary Stem Cells , 2010, Stem cells.

[30]  M. Paszek,et al.  Enforcing Order on Signaling , 2010, Science.

[31]  Mikala Egeblad,et al.  Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling , 2009, Cell.

[32]  Paolo P. Provenzano,et al.  The role of focal adhesion kinase in tumor initiation and progression , 2009, Cell adhesion & migration.

[33]  G. Smyth,et al.  ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. , 2009, Journal of immunological methods.

[34]  S. Fox,et al.  Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers , 2009, Nature Medicine.

[35]  G. Turashvili,et al.  A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability , 2008, Nature Medicine.

[36]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[37]  J. Brugge,et al.  Lumen formation during mammary epithelial morphogenesis: insights from in vitro and in vivo models , 2008, Cell cycle.

[38]  Patricia J. Keely,et al.  Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells , 2005, Biological Procedures Online.

[39]  M. Stampfer.  Cholera toxin stimulation of human mammary epithelial cells in culture , 1982, In Vitro.

[40]  C. Brisken,et al.  Stem Cells and the Stem Cell Niche in the Breast: An Integrated Hormonal and Developmental Perspective , 2007, Stem Cell Reviews.

[41]  Marie-Liesse Asselin-Labat,et al.  Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation , 2007, Nature Cell Biology.

[42]  Zena Werb,et al.  GATA-3 Maintains the Differentiation of the Luminal Cell Fate in the Mammary Gland , 2006, Cell.

[43]  Z. Werb,et al.  Comparative Mechanisms of Branching Morphogenesis in Diverse Systems , 2006, Journal of Mammary Gland Biology and Neoplasia.

[44]  Zlatko Trajanoski,et al.  CARMAweb: comprehensive R- and bioconductor-based web service for microarray data analysis , 2006, Nucleic Acids Res..

[45]  F. Bosman,et al.  Immunohistochemical Expression of Endothelial Markers CD31, CD34, von Willebrand Factor, and Fli-1 in Normal Human Tissues , 2006, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[46]  Haiyan I. Li,et al.  Purification and unique properties of mammary epithelial stem cells , 2006, Nature.

[47]  François Vaillant,et al.  Generation of a functional mammary gland from a single stem cell , 2006, Nature.

[48]  D. Proia,et al.  Reconstruction of human mammary tissues in a mouse model , 2006, Nature Protocols.

[49]  M. Sternlicht,et al.  Key stages in mammary gland development: The cues that regulate ductal branching morphogenesis , 2005, Breast Cancer Research.

[50]  Cynthia A. Reinhart-King,et al.  Tensional homeostasis and the malignant phenotype. , 2005, Cancer cell.

[51]  C. Eaves,et al.  Enzymatic dissociation and culture of normal human mammary tissue to detect progenitor activity. , 2005, Methods in molecular biology.

[52]  H. Parmar,et al.  Epithelial-stromal interactions in the mouse and human mammary gland in vivo. , 2004, Endocrine-related cancer.

[53]  A. Howell,et al.  Estrogen Responsiveness and Control of Normal Human Breast Proliferation , 2004, Journal of Mammary Gland Biology and Neoplasia.

[54]  Radhika Desai,et al.  ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix , 2003, The Journal of cell biology.

[55]  G. Dontu,et al.  In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. , 2003, Genes & development.

[56]  Mina J Bissell,et al.  Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. , 2002, Genes & development.

[57]  Claire Cameron,et al.  Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three–dimensional collagen gels , 1997, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[58]  J. Wolff,et al.  Forskolin stimulation of thyroid adenylate cyclase and cyclic 3',5'-adenosine monophosphate accumulation. , 1982, Endocrinology.

[59]  J. Mitchison.  Cell Biology , 1964, Nature.

[60]  R. K. Brown.  BIOPHYSICS , 1931 .