Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells - PubMed (original) (raw)

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells

Lixing Zhang et al. Cell Signal. 2017 Jan.

Abstract

Recent evidence suggests that mammary cells expressing R-spondin receptor and Wnt pathway regulator Lgr5, regarded as a stem cell marker in multiple tissues, might represent mammary stem cells (MaSCs). Whether L gr5 marks a multipotent subpopulation of Lin-CD24low/medCD49fhigh MaSCs remains controversial. To some extent the differing results reflect different assays used to assess properties of stemness, including lineage tracing in vivo, mammosphere culture, and mammary fat pad transplantation assays. To address this issue directly, we isolated Lgr5+ cells from mammary glands of Lgr5-lacZ mice and established organoids based on principles adapted from studies of Wnt-driven Lgr5+ cell populations in other organs. Mammary organoids were grown from single Lgr5+ mammary cells in Matrigel, the substratum of choice for intestinal organoids, and in a growth factor cocktail containing EGF, Wnt3a and R-spondin, designed to optimally activate the endogenous Wnt signaling program of stem cells. Colonies derived from single Lgr5+ cells manifest at least four distinct cell populations: Lgr5+ and Lgr5- basal cells and c-Kit+ and c-Kit- luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5- cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a "mini-breast" organoid.

Keywords: Lgr5(+); Mammary gland; Organoid; Stem cells.

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Conflict of interest statement

★ Disclosures: No potential conflicts are disclosed.

Figures

Fig. 1

Characterization of Lgr5+ cells from mouse mammary glands. (A,B) Isolation of mouse mammary Lgr5+ cells. Mouse mammary glands #3 and #4 from 8 to12 week old WT (A) or Lgr5-lacZ (B) virgin female mice were surgically resected, finely minced, collagenase treated, trypsin digested, and enriched for epithelial cells by negative selection. After staining with FDG, epithelial cells were analyzed for Lgr5 content by flow cytometry. Shown is one representative of 5 independent studies. (C) Percentage of Lgr5+ cells in Lgr5-EGFP-IRES-creERT2 mice. (D, E) To further characterize Lgr5+ cells, the Lin− population (TER119−), which represents 98.5% of the enriched epithelial cells (D), was fractionated by expression of the cell surface markers CD24 and CD49f by flow cytometry into three sub-populations (E): CD24low/medCD49fhigh basal cells, CD24+CD49flow/med luminal cells and CD24−CD49f− stromal cells. The majority of Lgr5+ (98.5%) cells were Lin−CD24low/medCD49fhigh basal cells (F) and constituted approximately 40% of all basal cells (G). (H) Percentage of Lgr5+ cells in the enriched epithelial population (as in B) and in the basal population (as in G). Data (mean ± SEM) are collated from 3 independent studies.

Fig. 2

Lgr5+ cells preferentially initiate organoid growth in vitro. Lgr5+ (A) and Lgr5− (B) populations were isolated as in Fig. 1B and equal numbers of cells were cultured in Matrigel with medium containing EGF, Wnt3a, andR-spondin1 (EWR) for 12 days. Organoid-forming efficiency (C) of the two populations, mean size of organoids (D), and extent of branching (E) were compared. Data (mean ± SD) are from 3 independent studies. In (F), organoid-forming efficiency was measured using Lgr5+ cells isolated from the basal cell population as in Fig. 1F. Data (mean ± SEM) are from 2 independent studies performed in quadruplicate. (G) Serial passage of organoids derived from Lgr5+ and Lgr5− cells. At the end of each 2-week passage, organoids were disrupted with trypsin, single cell suspensions were stained with FDG as in Fig. 1B, and Lgr5+ and Lgr5− populations were replated. Scale bar: 200 µm. Data (mean ± SEM) are from 2 independent studies, each with 4–6 replicates.

Fig. 3

Differentiation of organoids derived from single Lgr5+ cells. Lgr5+ cell-derived organoids cultured in Matrigel with EWR medium were heterogeneous: most of them, ~80%, were solid (A,D,G,J,M,P), while ~20% had a lumen (B,E,H,K,N,Q), as shown in H&E-stained sections (A, B). Sections of normal mammary ducts are shown for comparison (C,F,I,L,O). X-gal-stained Lgr5-lacZ+ cells line the outer cell layers of both solid and luminal colonies (D,E) and in normal ducts (F). In situ hybridization for Lgr5 RNA reveals basally distributed Lgr5+ cells in organoids (G, H) and in normal mammary ducts (I). Immunostaining for K14 and K8 shows the location of basal and luminal cells, respectively, in a typical solid organoid (J), organoid with lumen (K) and mouse mammary duct (L). Immunostaining for c-Kit shows the distribution of luminal progenitor cells in solid organoids (M), organoids with lumen (N) and normal mammary ducts (O). Nuclear and cytoplasmic β-catenin distribution in Lgr5+ cell-derived organoids is shown in (P, Q). Purple and brown double positive cells (arrow) indicate cells with nuclear β-catenin. Percentage of cells showing nuclear β-catenin in the outer and inner organoid cell layers is quantified in (R). Data (mean ± SD) are from 2 independent studies, examining 4 organoids per experiment. Scale bars: 20 µm.

Fig. 4

Ventral-dorsal gradient of Lgr5+ cell abundance in mammary glands correlates with organoid-forming ability of different zones. (A) Lgr5+ cell distribution in young adult mice. Whole-mount X-gal staining of mammary glands from 8 week old Lgr5-lacZ mice was performed as in [13]. Representative sections subsequently stained with fast red are shown. Subdivision of ventral (v), central (c) and dorsal (d) zone Lgr5+ epithelial cell fractions is shown schematically in (B), and quantification of LacZ+ cell content is shown in (C). Data (mean ± SEM) are from 3 glands. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Organoid-forming efficiency of epithelial cells from the different zones of the young adult mammary gland. The epithelial-enriched population isolated from each zone was plated in Matrigel containing EWR and quantified after 12–14 days (D). Data (mean ± SEM) are derived from 1 representative of 3 independent studies using 4–6 replicates per zone.

Fig. 5

Preferential expression of Lgr5 inventral ducts of the mammary gland during post-natal development, adulthood, and pregnancy. Whole-mount X-gal staining was performed as in Fig. 4 on mammary glands from 16 week old virgin WT mice (A), and Lgr5-lacZ transgenic mice of 5 weeks (B), 8 weeks (C) and 32 weeks (D) of age. The lower panels of figures A–D show 100× magnification of the ventral, central, and dorsal zones. Representative sections from each zone counterstained with fast red are shown in the lowest panel of figures A–D. X-gal staining was also performed on mammary glands from pregnant WT mice at pregnancy day 12 (E), and Lgr5-lacZ transgenic mice on pregnancy day 8.5 (F), day 12 (G) and day 14 (H). Lower panels show 100× magnification and representative sections counterstained with fast red.

Fig. 6

Hormone treatment alters organoid structure and induces squamous differentiation. Bright field images of organoids cultured in EWR medium (A)or EWR plus E2 and Pg (B). H&E-stained sections of organoids cultured in EWR (C) or in EWR plus hormones (D). Arrows in (C, D) identify organoid lumens. Organoid size (E) and lumen size (F) of treatment groups are shown. Data (mean ± SEM) are from 2 independent studies. (G–L) Immunofluorescent staining for K10 in organoids treated with EWR only (G, J) or EWR + E2 + Pg (H, I), and immunostaining for K14 and K8 (J, K, L). Scale bars: 20 µm. *P < 0.05, **P < 0.01.

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Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells - PubMed (original) (raw)