Quantification of regenerative potential in primary human mammary epithelial cells

Abstract

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.

Derivation of a robust mouse mammary organoid system for studying tissue dynamics

“…Overall, the efficiency of formation of branching organoids from freshly sorted single cells was higher than that previously reported for mouse organoids (∼4%) (Zhang et al, 2016a) or human TDLUlike structures (∼0.2%) (Linnemann et al, 2015;Sokol et al, 2016). Factors that likely contribute to these differences include the addition of FGF2 and heparin, the use of BME and the origin of cells (human versus mouse).…”

“…Development of mammary organoid models is currently an area of intense interest. Several recent studies on mouse or human breast epithelial cells have used advanced culture medium and 3D scaffolds for the formation of budding or TDLU-like structures that comprise luminal and basal cells layered in the correct orientation (Jardé et al, 2016;Linnemann et al, 2015;Panciera et al, 2016;Sokol et al, 2016;Zhang et al, 2016a,b). In this study, we have established conditions that enable robust formation of organoids from single sorted mouse basal and luminal epithelial cells, with high efficiency and reproducibility.…”

Modeling Development and Disease with Organoids

“…Basal and luminal markers were expressed at correct positions, and the ducts displayed contractility. Thus, the organoids resembled terminal ductal-lobular units, the functional units of the mammary gland (Linnemann et al, 2015). Since Wnt signals and Lgr5 have been implied in mammary stem cell biology (Plaks et al, 2013;Rios et al, 2014), it will be of interest to test the effects of the addition of Wnt/R-spondin to these cultures.…”

Complexity galore: 3D cultures, biomechanics and systems medicine at the eighth ENBDC workshop “Methods in Mammary Gland Development and Cancer”

“…She discussed a novel 3D culture method for normal human breast epithelium [12]. Human breast epithelial cells obtained from reduction mammoplasties were cultured as organoids in floating collagen I gels in medium containing bovine pituitary extract and forskolin.…”