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.
Highlights
The mammary gland (MG) develops from the anlage, a cluster of specified ectodermal cells forming a rudimentary ductal tree before birth (Sternlicht, 2006)
Identification of culture conditions that promote generation of terminal ductal-lobular units (TDLUs)-like structures To recapitulate morphogenesis in a 3D-culture system, we chose collagen type I as a substrate, because it constitutes a main component of extracellular matrix in the human MG and provides defined properties that can be modified to model different microenvironments
10-12 days, freshly isolated single-cell suspensions of human mammary epithelial cells (HMECs) cultured in floating collagen gels gave rise to multicellular structures that were subdivided into three types of branched (TDLU-like, thin, star) and three types of non-branched structures
Summary
The mammary gland (MG) develops from the anlage, a cluster of specified ectodermal cells forming a rudimentary ductal tree before birth (Sternlicht, 2006). Puberty induces outgrowth into an expansive network of ducts, draining the milk-producing, terminal ductal-lobular units (TDLUs) (Brisken and O’Malley, 2010). Defining the molecular identity of MaSCs remains an active area of investigation
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