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. 2013 Jan 31;3(1):70-8.
doi: 10.1016/j.celrep.2012.12.017. Epub 2013 Jan 24.

Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis

Vicki Plaks  1 , Audrey Brenot, Devon A Lawson, Jelena R Linnemann, Eline C Van Kappel, Karren C Wong, Frederic de Sauvage, Ophir D Klein, Zena Werb
Affiliations

Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis

Vicki Plaks et al. Cell Rep. .

Abstract

Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5(+)) mammary epithelial cells (MECs). We found that Lgr5(+) cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs), are exceptionally efficient in reconstituting MGs from single cells, and exhibit regenerative capacity in serial transplantations. Loss-of-function and depletion experiments of Lgr5(+) cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis.

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Figures

Figure 1
Figure 1. Lgr5 Expression is Restricted to a Rare Subpopulation of Cytokeratin 14+, Lin−CD24+CD49fhigh Mammary Basal Cells
(A) The expression of Lgr5 was examined in cryosections from 7-week-old Lgr5-EGFP MGs with an anti-GFP antibody (green). Carmine stain of a representative MG whole mount demonstrates that Lgr5+ ducts are located to the nipple area, but not to the invading front. Around the lymph node there are some positive and negative ducts. LN, lymph node. (B) Cryosections co-stained with anti-GFP and anti-K14. Lgr5+ cells (green) are a located to the supra-basal layer of the ducts and are a subpopulation of the myoepithelial K14+ cells (red). (C) MGs isolated from Lgr5-EGFP mice and analyzed by flow cytometry, for the expression of the cell surface markers Ter119, CD45, CD31 (Lin), CD24 and CD49f. All Lgr5+ cells (GFP+) were part of the Lin−CD24+CD49fhigh cells (stem cell-enriched population). Lgr5+ cells are 0.3% of total mammary cells and 2.5% of Lin−CD24+CD49fhighbasal cells. GFP+ cells within the luminal population are 0.009% of total. (D) Summary of flow cytometry data in Figure 1C, Lgr5+ cells in 7.5-week-old pubertal female mice.% of Lgr5+ cells of total (n=14) and of Lin−CD24+CD49fhighbasal cells (n=7). Also see Figure S1. (E) Real-time, quantitative PCR analysis of the Lgr5+ cell population (relative to Lgr5− mammary cells) revealed they are high for basal but not luminal markers. PR, progesterone receptor; ERα, estrogen receptor α. See also Table S1. Error bars represent SE.
Figure 2
Figure 2. Within the Lin−CD24+CD49fhigh Basal Population, Lgr5+ Cells are Highly Potent in Generating Functional Mammary Outgrowths
(A) Lgr5+ (GFP+) and non-expressing (GFP−) cells from Lgr5-EGFP were isolated by flow cytometry from the Lin−CD24+CD49fhigh basal population and injected (10, 50 or 100 cells) into cleared mammary fat pads. Outgrowths were analyzed 6 weeks post-transplantation. (B) Transplanted basal Lgr5+ cells have higher numbers of outgrowths compared to the basal Lgr5-cells. Data pooled from 3 different experiments. (C) Whole mount carmine-stained representative outgrowths show that 10 basal Lgr5+ cells are able to reconstitute a full MG vs. no outgrowth for basal Lgr5-transplanted cells. (D) Mice transplanted with 10 Lgr5+ cells were mated with males and their MGs analyzed on day 18.5 (E18.5) of pregnancy. (E) Whole mount Carmine-stained mammary epithelial outgrowths from E18.5 pregnant female mice transplanted with 10 basal Lgr5+ cells that underwent full lobuloalveolar differentiation (basal Lgr5+), comparable to the endogenous epithelium in MG #3 of the recipient mouse (upper panels). MG sections from the same mice stained positive for the milk protein, β-casein (lower panels; brown). See also Figure S2 and Table S2.
Figure 3
Figure 3. Lgr5+ cells can Regenerate a Mammary Gland from a Single Cell and Maintain Regenerative Potential through Serial Transplantations
(A) Single mammary Lgr5+ (GFP+) cells from Lgr5-EGFP crossed into the Life Act-RFP mice were isolated by flow cytometry into 96-well plates and transplanted into cleared mammary fat pads. Outgrowths were analyzed at 8 weeks post-transplantation. (B) From transplants of single adult mammary Lgr5+ cells in 54 mammary glands, 13 mammary outgrowths were observed. (C) A representative RFP+ mammary outgrowth from a single Lgr5+ cell, exhibiting a full epithelial tree (left) with ductal structures at higher magnification of boxed area (right). (D) Outgrowths from single Lgr5+ cells differentiate into the myoepithelial (K14+ in red) and luminal (K8+ in green) lineages (left). Boxed area magnified (right). See also Figure S3. (E) Mammary outgrowth from 2 mice transplanted with 100 Lgr5+ cells (isolated from Lgr5-EGFP crossed into the LifeAct-RFP mice) were collected and transplanted into 10 mice each for secondary and the same for tertiary outgrowths. (F,G) Lgr5+ outgrowths retain their regenerative potential through secondary (F) and tertiary (G) transplants. RFP images are representative of the mammary outgrowths.
Figure 4
Figure 4. Depletion Experiments Demonstrate that Lgr5+ Cells are Necessary for Mammary Gland Epithelial Reconstitution
(A) Depletion of Lgr5+ cells was achieved utilizing Lgr5-DTR:GFP crossed into actin-RFP mice, injected with 50ng/g BW DTx, analyzed 24h post DTx i.p. (Lgr5+ cells are 0.1% of total dissociated mammary cells versus 0% in DTx- injected mice). (B) Isolated primary MECs of Lgr5-DTR:GFP mice or WT littermates transplanted into contralateral pre-cleared mammary fat pads with or without DTx administration. MGs collected 3 weeks post-transplantation had significantly impaired outgrowths in the Lgr5-DTR:GFP transplants vs. the WT controls. (C) To assess the growth potential of the Lgr5-DTR:GFP and control littermate, mice transplanted with the same cells as in (B) but not treated with DTx reveal no difference between the two contralateral sides. (D) Outgrowth area for Lgr5-DTR:GFP epithelial transplants (including impaired ducts) relative to the contralateral WT transplants is significantly reduced in DTx- treated mice (*p= 0.006). Bars represent SE. See also Figure S4.
Figure 5
Figure 5. Depletion of Lgr5+ Cells During Pubertal Development Results in Impaired Ductal Invasion and Terminal End Bud Formation
(A) Carmine-stained MG of 4.5-week-old Lgr5-DTR:GFP mice (n=6) or WT littermates (n=4) that were i.p. injected with DTx demonstrate significantly reduced ductal invasion in the Lgr5-DTR:GFP mice. (B) Quantification of data presented in (A). (C) Depletion of Lgr5+ cells from Lgr5-DTR:GFP mice resulted in significant reduction in the number of TEBs per MG versus WT littermates. (D) Quantification of data presented in (C). (E) Whole mounts of 5-week-old Lgr5-EGFP-IRES-creERT2/Rosa-Tomato mice one week past start of Tamoxifen (TAM) induction, indicated that Lgr5+ cell progeny are close to the nipple area (left) and, according to their localization and shape, mark myoepithelial cells (middle, enlargement of red boxed area in left) and not TEBs in the invading front (Carmine-stained tissue on right). Error bars represent SE. See also Figure S5.
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