Essential role for a novel population of binucleated mammary epithelial cells in lactation

Rios, Anne C.; Fu, Nai Yang; Jamieson, Paul; Pal, Bhupinder; Whitehead, Lachlan; Nicholas, Kevin R.; Lindeman, Geoffrey J.; Visvader, Jane E.

doi:10.1038/ncomms11400

Cited by 90 publications

(106 citation statements)

References 39 publications

(58 reference statements)

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“…The following day, organoids were washed and incubated in 80% glycerol for 1 h before 3D imaging using a SP8 confocal microscope. 3D rendering was performed using the Imaris software as described previously (Rios et al, 2014(Rios et al, , 2016. Confetti-fluorescent living cultures were imaged as described above, and scored using the 3D visualization module of Imaris.…”

Section: Organoid Culturementioning

confidence: 99%

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

et al. 2016

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Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited options for studying mouse mammary gland formation in vitro. Here, we have built upon previously described culture assays to define culture conditions that enable the efficient generation of clonal organoid structures from single sorted basal mammary epithelial cells (MECs). Analysis of Confetti-reporter mice revealed the formation of uni-colored structures and thus the clonal nature of these organoids. Highresolution 3D imaging demonstrated that basal cell-derived complex organoids comprised an inner compartment of polarized luminal cells with milk-producing capacity and an outer network of elongated myoepithelial cells. Conversely, structures generated from luminal MECs rarely contained basal/myoepithelial cells. Moreover, flow cytometry and 3D microscopy of organoids generated from lineagespecific reporter mice established the bipotent capacity of basal cells and the restricted potential of luminal cells. In summary, we describe optimized in vitro conditions for the efficient generation of mouse mammary organoids that recapitulate features of mammary tissue architecture and function, and can be applied to understand tissue dynamics and cell-fate decisions.

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Section: Organoid Culturementioning

confidence: 99%

“…Basal cells could form bi-lineage organoids within 2 weeks at a frequency of 20%. Characterization of these structures using high-resolution 3D imaging technology (Rios et al, 2014(Rios et al, , 2016 and lineage-specific mouse reporter models has provided novel insights into the cellular dynamics of organoid formation.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…As a result, immunolabelling and fluorescence imaging of mammary tissue has traditionally been performed using thin tissue sections with assumptions about the architectural context and uniformity of a particular 2D plane. Whilst 3D imaging has recently been used to investigate mammary stem cell dynamics [2–4] and binucleated cells in lactational alveoli [5], these studies have relied on tissue microdissection [2, 5] or enzymatic digestion [3]. Consequently, visualisation of the mammary epithelial tree at single-cell resolution within its native stroma remains a fundamental challenge in mammary gland research.…”

Section: Introductionmentioning

confidence: 99%

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods

et al. 2016

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BackgroundHigh-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated.MethodsMultiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed.ResultsVarying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue.ConclusionsThe utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0754-9) contains supplementary material, which is available to authorized users.

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“…Sequential thin optical sections through a single alveolus (Fig. C) show that CD45+ cells appear to intercalate between the overlying basal cells and the luminal cells, which are frequently binucleated in the lactating mammary gland . A comparison of cells in the virgin ducts with those in the alveoli (compare Fig.…”

Section: Resultsmentioning

confidence: 96%

Dynamic architectural interplay between leucocytes and mammary epithelial cells

et al. 2019

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The adult mammary gland undergoes dynamic changes during puberty and the postnatal developmental cycle. The mammary epithelium is composed of a bilayer of outer basal, or myoepithelial, cells and inner luminal cells, the latter lineage giving rise to the milk-producing alveolar cells during pregnancy. These luminal alveolar cells undergo Stat3-mediated programmed cell death following the cessation of lactation. It is established that immune cells in the microenvironment of the gland have a role to play both in the ductal outgrowth during puberty and in the removal of dead cells and remodelling of the stroma during the process of postlactational regression. However, most studies have focussed on the role of the stromal immune cell compartment or have quantified immune cell populations in tissue extracts. Our recent development of protocols for deep imaging of the mammary gland in three dimensions (3D) has enabled the architectural relationship between immune cells and the epithelium to be examined in detail, and we have discovered a surprisingly dynamic relationship between the basal epithelium and leucocytes. Furthermore, we have observed morphological changes in the myoepithelial cells, as involution progresses, which were not revealed by previous work in 2D tissue sections and whole tissue. This dynamic architecture suggests a role for myoepithelial cells in the orderly progression of involution. We conclude that deep imaging of mammary gland and other tissues is essential for analysing complex interactions between cellular compartments.Abbreviations 2D, two dimensions; 3D, three dimensions; CD, cluster of differentiation; DAPI, 4 0 ,6-diamidino-2-phenylindole; DCIS, ductal carcinoma in situ; DCs, dendritic cells; DNA, deoxyribonucleic acid; GPI, glycosylphosphatidylinositol; H&E, haematoxylin and eosin; IgA, immunoglobulin A; K5, keratin 5; LIF, leukaemia inhibitory factor; MHCII, major histocompatibility complex class II; MIP, maximum intensity projection; MMPs, matrix metalloproteinases; NF-jB, nuclear factor kappa-light-chain-enhancer of activated B cells; SEMA, semaphorin; SMA, smooth muscle actin isoform a-actin; Stat, signal transducer and activator of transcription; TEBs, terminal end buds; Th, T helper; VEGF, vascular endothelial growth factor.

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Essential role for a novel population of binucleated mammary epithelial cells in lactation

Cited by 90 publications

References 39 publications

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

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

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods

Dynamic architectural interplay between leucocytes and mammary epithelial cells

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