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.

Wnt signaling has been implicated in different stages of mammary development as well as in mammary oncogenesis (). The Wnt coreceptor Lrp5 has been described as a marker of mammary stem cells (), and secreted Wnt proteins are proposed as important self-renewal factors for MG stem cells (). Lgr5, a downstream target of Wnt, was identified as a marker of adult stem cell populations in the small intestine, colon (), stomach (), and hair follicle (), organs that undergo extensive postnatal regeneration. Recently, lineage-tracing experiments revealed that Lgr5stem/progenitor cells also contribute to nephron formation during kidney development (). Here, we unmask the regenerative potential of a rare Lgr5-expressing (Lgr5) mammary cell population and its indispensable contribution to pubertal mammary development.

The Wnt receptor, Lrp5, is expressed by mouse mammary stem cells and is required to maintain the basal lineage.

Wnt-3, a gene activated by proviral insertion in mouse mammary tumors, is homologous to int-1/Wnt-1 and is normally expressed in mouse embryos and adult brain.

Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.

Lef1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland.

Adult stem cells are characterized by their ability to both self-renew and to differentiate into specialized cells. Unraveling the hierarchy of mammary stem and progenitor cells has been of great interest because the mammary gland (MG) undergoes extensive tissue expansion and remodeling at various phases throughout adult life. Moreover, deciphering the stem cell players contributing to normal mammary development is key to understanding subsequent pathologies, such as cancer transformation. During pubertal development, which happens between 3 and 8 weeks of age in mice, the mammary epithelium undergoes glandular expansion. This yields a branching network of ducts composed of two primary epithelial cell lineages: myoepithelial/basal, and luminal. During pregnancy, the epithelium goes through additional lobuloalveolar differentiation to allow lactation (). The MG can be regenerated efficiently by transplanting mammary epithelial cells (MECs) into cleared mammary fat pads. Serial transplantation and limiting dilution assays of primary cultures derived from clonal outgrowths have pointed to the existence of a rare subset of mammary cells that function as stem cells and reconstitute functional MGs (). This basal population, which includes mammary stem cells, is characterized by the surface antigen profile LinCD24CD29or LinCD24CD49f) and is enriched in Wnt-responsive cells ().

(C) Flow cytometry analysis of MGs from 5-week-old Lgr5-EGFP-IRES-creERT2/Rosa-Tomato mice, 1 week after the start of TAM induction. Note that most of the Tomato+ progeny still maintain their Lgr5 expression (lower left). When Lgr5+ progeny is backgated on mammary subsets (upper right), it is found to localize exclusively to the basal population, which includes myoepithelial cells (lower right plot).

(B) MG #3 also demonstrates how Lgr5+cell progeny (marked area on left) is localized to a very restricted area of the entire ductal tree (marked area on right) and does not localize to TEBs.

(A) By whole mount fluorescence analysis, Lgr5+ cell progeny are localized to myoepithelial cells at 2 and 3 weeks post the start of Tamoxifen (TAM) induction (red; images on right are enlargement of boxed are on left), according to their localization and shape. TAM induction was started at 4 weeks of age so that 2 and 3 weeks induction corresponded to 6 and 7 weeks of age, respectively. Note that over-exposed images on left confirm that TEBs are negative for red fluorescence.

(E) Whole mounts of 5-week-old Lgr5-EGFP-IRES-creERT2/Rosa-Tomato mice 1 week past start of Tamoxifen (TAM) induction indicated that Lgr5 + cell progeny is close to the nipple area (left) and, according to its 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).

(C) Depletion of Lgr5 + cells from Lgr5-DTR:GFP mice resulted in significant reduction in the number of TEBs per MG versus WT littermates. Arrows indicate the TEBs.

(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 demonstrates significantly reduced ductal invasion in the Lgr5-DTR:GFP mice.

To complement the results above, we examined the role of Lgr5cells in postnatal MG organogenesis, in a more physiological setting, by injecting DTx to pubertal mice that were either Lgr5-DTR:GFP or WT littermates ( Figure 5 ). Depletion of Lgr5cells during pubertal MG development resulted in impaired ductal invasion ( Figures 5 A and 5B) and, interestingly, also in a significant reduction in the number of terminal end buds (TEBs) at the epithelial invading front ( Figures 5 C and 5D), even though Lgr5cells ( Figure 1 A) and their lineage-specific progeny ( Figures 5 E and S5 ) are absent from the TEBs. In this context, although Lgr4 has been shown to play a minor role in MG development (), Lgr4+ cells were not interchangeable with Lgr5cells because a significant phenotype was observed upon Lgr5cell depletion. These data show that under normal physiology, although all other cells (including additional progenitor cells) were not depleted, the presence of Lgr5cells is necessary for MG pubertal development and reinforce the depletion results in the transplantation setting.

(C) In vitro mammary sphere-forming assay using 10 4 primary mammary epithelial cells from Lgr5-DTR:GFP mice (the same number of cells used for the in vivo depletion experiments) show that cells cultured in the presence of DTx are unable to form spheres, as analyzed on day 8 of culture. Data are summarized from 2 independent experiments each with cells from 4 mice and 2-4 wells per condition. Bars represent SE ( ∗ p = 0.003). Representative images of spheres are included.

(B) Carmine stains of the PBS-or DTx-injected recipient non-transplanted MGs #3 of 8-week-old nude mice exhibit normal ductal structures, indicating that DTx is not toxic to endogenous WT mature mammary epithelial tissue.

(A) Outgrowths from additional recipient mice to those presented in Figure 4 B, transplanted with epithelial cells from Lgr5-DTR:GFP x actin-RFP or actin-RFP littermate into contralateral pre-cleared mammary fat pads with DTx administration. Lower panels are higher magnifications of the boxed areas in the corresponding upper panels. These data demonstrate runted and fewer ducts in outgrowths of mice administered with DTx. The 60% decrease in ductal area shown in Figure 4 D for the Lgr5-DTR:GFP transplants treated with DTx includes these impaired structures, although they do not form proper ductal structures.

Effects of Diphtheria Toxin-Mediated Depletion of Lgr5+ Cells on Mammary Outgrowths and In Vitro Sphere Formation, Related to Figure 4

(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.

(B) Isolated primary MECs of Lgr5-DTR:GFP mice or WT littermates transplanted into contralateral precleared mammary fat pads with or without DTx administration. MGs collected 3 weeks posttransplantation had significantly impaired outgrowths in the Lgr5-DTR:GFP transplants versus the WT controls.

(A) Depletion of Lgr5 + cells was achieved utilizing Lgr5-DTR:GFP crossed into actin-RFP mice, injected with 50 ng/g BW DTx, analyzed 24 hr post-DTx i.p. (Lgr5 + cells are 0.1% of total dissociated mammary cells versus 0% in DTx-injected mice).

To determine whether Lgr5cells are not only sufficient but also necessary for postnatal MG organogenesis, we used the Lgr5-DTR:GFP mice to deplete Lgr5cells following administration of diphtheria toxin (DTx) ( Figure 4 A). This mouse model was used previously to demonstrate the dispensability of intestinal Lgr5cells under steady-state conditions (). However, depletion of Lgr5cells from transplanted MECs immediately posttransplantation impaired the outgrowth of Lgr5-DTR:GFP donor epithelium, compared to the contralateral MG transplanted with WT MECs ( Figure 4 B). As an additional control, we found that the majority of MECs from Lgr5-DTR:GFP and WT mice not treated with DTx (i.e., in the presence of Lgr5cells) were able to reconstitute mammary outgrowth ( Figure 4 C). Uncleared, endogenous mammary tissue from the WT recipient mice was not affected by DTx administration ( Figure S4 B). The total outgrowth area for Lgr5-DTR:GFP epithelial transplants (including impaired ducts, as shown in Figure S4 A) was also significantly reduced in DTx-treated mice relative to the contralateral WT transplants ( Figure 4 D). These experiments indicate that, although all other epithelial cells were not depleted, the absence of Lgr5cells was detrimental to adequate MG reconstitution. This protocol allowed targeted MG Lgr5cell depletion because the recipient mice do not carry the Lgr5-DTR:GFP transgene. Mammosphere-forming assays in culture confirmed the indispensability of Lgr5cells ( Figure S4 C).

Additional examples of mammary ductal outgrowths from mammary transplants of single Lgr5+ cells isolated from LifeAct-RFP+ mammary glands and transplanted into cleared fat pads from unlabeled female nude mice.

(F and G) Lgr5 + outgrowths retain their regenerative potential through secondary (F) and tertiary (G) transplants. RFP images are representative of the mammary outgrowths.

(E) Mammary outgrowths from two mice transplanted with 100 Lgr5 + cells (isolated from Lgr5-EGFP crossed into the LifeAct-RFP mice) were collected and retransplanted into ten mice each for secondary and the same for tertiary outgrowths.

(D) Outgrowths from single Lgr5cells differentiate into the myoepithelial (K14in red) and luminal (K8in green) lineages (left). Boxed area magnified (right). See also Figure S3

(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).

(A) Single mammary Lgr5 + (GFP + ) cells from Lgr5-EGFP crossed into the LifeAct-RFP mice were isolated by flow cytometry into 96-well plates and transplanted into cleared mammary fat pads. Outgrowths were analyzed at 8 weeks posttransplantation.

Because Lgr5cells within the basal cell population were highly efficient in regenerating a full MG in limiting dilution experiments, we tested them for classical stem cell characteristics of multipotency and self-renewal. First, we assessed their ability to regenerate fully differentiated MGs from single cells ( Figure 3 A). We observed that 13 outgrowths were generated from 54 single Lgr5transplanted cells ( Figure 3 B), demonstrating that 24% of Lgr5single cells were able to regenerate a full MG equivalent to 1 MRU per 4.2 Lgr5cells. These results are similar to those of the limiting dilution experiments ( Figure 2 ). On close examination, we observed substantial epithelial outgrowth in the mammary fat pads ( Figures 3 C and S3 ) and demonstrated that these single transplanted Lgr5cells were multipotent because they were able to differentiate into both mammary epithelial lineages (myoepithelial/basal K14and luminal K8cells) ( Figure 3 D). In addition, when we serially transplanted epithelial outgrowths from primary transplants of Lgr5cells ( Figure 3 E), the Lgr5outgrowths retained their regenerative potential through secondary and tertiary transplants, demonstrating a long-term, regenerative potential ( Figures 3 F and 3G).

(C) Analysis of the Delta Ct average fold change values relative to the basal Lgr5-cells, summarizing data from the 3 experiments, reveals that the Lgr5+ cells are overall higher for basal markers (red bars- K5, p63) and lower for luminal markers (blue bars- K8, K18, ERα, Muc1, β-casein) within the basal population. These results summarize data from 3 experiments. The genes that exhibited the biggest and significant changes across 3 experiments were p63 and β-casein (p = 0.01). Interestingly, the basal marker p63, which is also necessary for the normal development of human mammary glands, was found to be a positive prognostic factor in endocrine-treated ER positive breast cancer (), thereby indicating that Lgr5+ cells can serve as cancer initiating cells. The observation that β-casein is downregulated supports the notion that the Lgr5+ cells are more stem-like and therefore, less differentiated than the other basal cells. Bars represent standard error.

(B) All single basal Lgr5- cells versus basal Lgr5+ cells represented in a bar graph according to their relative expression levels reveal that all cells sorted as low for GFP within the basal population display relatively low Lgr5 expression levels. Cells that were sorted as high for GFP show higher levels of Lgr5 expression in most cells (10/14) but also contain cells with expression levels similar to the Lgr5- cells (4/14). This could also explain why not all Lgr5+ sorted cells were able to generate mammary outgrowths, as described in Figures 2 B and 3 B. The bar graph is based on the data presented in (A).

(A) Heatmap of unsupervised clustering of the expression data of the 29 genes tested [markers of mammary lineage differentiation, stem cell and signaling pathways (Wnt, Notch), pluripotency and epithelial-to-mesenchymal transition] reveals that within the basal population, sorted basal Lgr5+ cells (n = 8/14), cluster together versus other basal Lgr5- cells (n = 10/11). Cells were considered Lgr5+ based on higher GFP expression within the basal population. Orange box demarcates the clustering of Lgr5+cells. The color scale represents the centered and scaled log transformed expression values, and gray represents failed reactions. The heatmap is a representative of 3 experiments; each tested 25-60 single cells.

Single cell PCR analysis was performed on cells sorted using the flow cytometry methodology described in Figures 2 A and 3 A. + or - indicate GFP expression within the basal population, as sorted by flow cytometry.

Single basal Lgr5+ cells versus LinCD24CD49fbasal Lgr5- cells were characterized to elucidate whether the different functional mammary reconstitution abilities of the two subsets are based on differences in gene expression, similar to the rationale of analyzing intestinal Lgr5+ cell (). Therefore, the single cell expression analysis explored only a few genes and focused on markers related to stem/progenitor capacity, rather than on signaling molecules.

(E) Whole-mount carmine-stained mammary epithelial outgrowths from E18.5 pregnant female mice transplanted with ten 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).

(D) Mice transplanted with ten Lgr5 + cells were mated with males, and their MGs were analyzed on day 18.5 (E18.5) of pregnancy.

(C) Whole-mount carmine-stained representative outgrowths show that ten basal Lgr5 + cells are able to reconstitute a full MG versus no outgrowth for basal Lgr5-transplanted cells.

(B) Transplanted basal Lgr5 + cells have higher numbers of outgrowths compared to the basal Lgr5 − cells. Data are pooled from three different experiments.

(A) Lgr5 + (GFP + ) and nonexpressing (GFP − ) cells from Lgr5-EGFP were isolated by flow cytometry from the Lin − CD24 + CD49f high basal population and injected (10, 50, or 100 cells) into cleared mammary fat pads. Outgrowths were analyzed 6 weeks posttransplantation.

The analysis described above revealed that Lgr5cells are a subset of the LinCD24CD49fbasal cells previously reported to include stem cells (). To assess MG reconstitution competence, we challenged the Lgr5cells for mammary regeneration and compared them to Lgr5-negative (Lgr5) basal cells in limiting dilution experiments ( Figure 2 A). In these experiments, we transplanted 10, 50, and 100 Lgr5versus Lgr5basal cells into cleared fat pads. The number of cells transplanted was chosen on the lower range to increase the stringency of the assay, focus on a small subset of basal cells, and avoid false negatives owing to Lgr5cells that express low levels of GFP and could therefore be sorted into the Lgr5group. We found that within the basal population, Lgr5cells generated MGs far more efficiently than did basal Lgr5cells. On average, 27% (±5% SE) of Lgr5cells were able to regenerate a full MG, within the 10–100 cell range, or 1 MRU per 3.7 Lgr5cells ( Figures 2 B and 2C). We then tested functionality upon pregnancy ( Figure 2 D) and found that these outgrowths were able to undergo full lactational lobuloalveolar differentiation and express the milk protein, β-casein ( Figure 2 E). Characterization of single basal Lgr5cells versus basal Lgr5cells revealed that the different functional mammary reconstitution abilities of the two subsets are based on differences in gene expression of lineage differentiation, stem cell, and pluripotency markers, demonstrating that these populations are distinct ( Figure S2 ).

Flow cytometry of mammary glands (MGs) from 27-week-old mice, shows that the Lgr5+ cells were still mostly located within the Lin - CD24 + CD49f high basal cell population. Within the basal cells, Lgr5+ cells are restricted to a CD24 high CD49f high population (Lgr5+ cells are 1.6% of total mammary cells and 16.3% of Lin - CD24 + CD49f high basal cells; GFP+ cells are 0.003% of total within the luminal population).

(E) Real-time, quantitative PCR analysis of the Lgr5cell population (relative to Lgr5mammary cells) revealed that they are high for basal but not luminal markers. PR, progesterone receptor; ERα, estrogen receptor α. See also Table S1

(D) Summary of flow cytometry data in Figure 1 C, Lgr5cells in 7.5-week-old pubertal female mice, percentage (%) of Lgr5cells of total (n = 14), and of LinCD24CD49fbasal cells (n = 7). See also Figure S1

(C) MGs were 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. Lgr5 + cells (GFP + ) were part of the Lin − CD24 + CD49f high cells (stem cell-enriched population). Lgr5 + cells are 0.3% of total mammary cells and 2.5% of Lin − CD24 + CD49f high basal cells. GFP + cells within the luminal population are 0.009% of total.

(B) Cryosections costained with anti-GFP and anti-K14. Lgr5 + cells (green) are located to the suprabasal layer of the ducts and are a subpopulation of the myoepithelial K14 + cells (red).

(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 (LN), there are some positive and negative ducts.

To identify cells that express Lgr5 in the MG, we used the Lgr5 knockin mouse model in which EGFP reporter gene expression is driven by the endogenous Lgr5 regulatory sequences (). In adult pubertal MGs, only 14% (±2% SE) of ducts had Lgr5cells, and they were all localized to the nipple side (taking the lymph node as a point of reference), as previously illustrated by. The nipple is where the fetal epidermis initially invaginates into the mammary fat pad and is the origin growth point of the mammary epithelium ( Figure 1 A). Lgr5cells were a subset of cytokeratin 14-positive (K14) cells and were localized to the suprabasal position ( Figure 1 B), similar to that previously described for mammary stem cells by. In MGs, adult stem cells have been defined by flow cytometry as a rare subset of LinCD24CD29) or LinCD24CD49fbasal cells (), and a subpopulation of such cells exhibits the capacity to regenerate an entire MG in vivo. The vast majority of Lgr5cells were basal, LinCD24CD49f Figure 1 C and Figure S1 ) and were quite rare, comprising 0.26% (one Lgr5cell per 386 cells) of total dissociated cells in pubertal MGs ( Figure 1 D). Previous studies have estimated the frequency of mammary stem cells or mammary repopulating units (MRUs) from adult virgin mouse MG to be 1 per 1,400 dissociated cells (for FVB background;); in contrast, 3%–7% of cells in intestinal crypts express Lgr5 (). In pubertal glands, among the mammary basal cells, only 6% were Lgr5 Figure 1 D); this was corroborated by the expression profile of Lgr5cells, which showed high levels of basal but low levels of luminal epithelial markers ( Figure 1 E).

Discussion

+ cells have been identified as adult stem cells in the small intestine, colon ( Barker et al., 2007 Barker N.

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Clevers H. Very long-term self-renewal of small intestine, colon, and hair follicles from cycling Lgr5+ve stem cells. + cells are also adult stem cells in the MG. By transplantation assays, we demonstrated that most Lgr5+ cells are a subset of the basal population previously shown to include the mammary stem cells, exhibiting superior reconstitution capabilities as compared to other cells within that population and are also extremely efficient in regenerating a MG from a single cell. The reconstituted MG epithelial tree was also functional because it was able to undergo adequate differentiation during pregnancy and produce a milk protein. Lgr5+ cells were multipotent and maintain regenerative potential in serial transplantations and, therefore, sufficient for postnatal MG organogenesis. They were also necessary for MG organogenesis as shown in depletion assays in both transplantation and physiological settings. Classically, stem cells are characterized by their ability to self-renew as well as to differentiate into specialized cells. According to these criteria, Lgr5cells have been identified as adult stem cells in the small intestine, colon (), stomach (), and hair follicle (). Our study now shows that Lgr5cells are also adult stem cells in the MG. By transplantation assays, we demonstrated that most Lgr5cells are a subset of the basal population previously shown to include the mammary stem cells, exhibiting superior reconstitution capabilities as compared to other cells within that population and are also extremely efficient in regenerating a MG from a single cell. The reconstituted MG epithelial tree was also functional because it was able to undergo adequate differentiation during pregnancy and produce a milk protein. Lgr5cells were multipotent and maintain regenerative potential in serial transplantations and, therefore, sufficient for postnatal MG organogenesis. They were also necessary for MG organogenesis as shown in depletion assays in both transplantation and physiological settings.

−CD24+CD29high population of mammary cells ( Shackleton et al., 2006 Shackleton M.

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Wahl G.M. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. The frequency of MRUs was previously estimated to be between 1 MRU per 8–17 cells using transplants of single cells or 1 per 64 cells within the LinCD24CD29population of mammary cells () or 1 per 60 cells (for FVB background) and 1 per 90 cells (for C57BL/6 background) within the LinCD24CD49fcells () in limiting dilution experiments. More recently, the stem cell frequency within the adult LinCD24CD49fpopulation was estimated as 1 per 50 cells when coinjected with Matrigel (). The reconstitution capabilities of one per four cells that we observed are remarkable, bringing us closer to obtaining a homogeneous population of MRUs.

Shackleton et al., 2006 Shackleton M.

Vaillant F.

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Asselin-Labat M.L.

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Visvader J.E. Generation of a functional mammary gland from a single stem cell. Stingl et al., 2006 Stingl J.

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Blanpain C. Distinct stem cells contribute to mammary gland development and maintenance. +cells, which was reinforced in a recent study by de Visser et al. (2012) de Visser K.E.

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Blanpain C. Distinct stem cells contribute to mammary gland development and maintenance. + cell is sufficient to regenerate a complete mammary epithelium and differentiates into both myoepithelial and luminal cells. The transplant assays might therefore uncover a regenerative potential of Lgr5+ cells that would be inhibited during MG pubertal development. However, in all the previous studies, depletion of a specific cell population in the presence of all the other cells was not attempted. We now have demonstrated that, in the MG, the unique approach of specific Lgr5+ cell depletion resulted in significantly impaired organogenesis, indicating that Lgr5+ cells are required during both regeneration from transplanted MECs but also, and more importantly, during physiological pubertal development. Although previous transplant experiments suggested a common progenitor for both major mammary epithelial lineages (myoepithelial/basal and luminal) (), a recent study that utilized lineage-tracing assays pointed to two different progenitors for these lineages as early as birth () and, therefore, suggested a more restricted fate for the Lgr5cells, which was reinforced in a recent study byand also in our study. These data point to important differences between lineage-tracing and transplantation techniques. Indeed, individual stem cells can have different roles under physiological, homeostatic conditions visualized by lineage tracing (), compared to when they are challenged to regenerate an entire organ in the transplant assays (). Thus, lineage-tracing experiments using an Lgr5-CreER line show that Lgr5cells give rise only to myoepithelial cells in pubertal MGs (), whereas our transplant experiments demonstrated that a single Lgr5cell is sufficient to regenerate a complete mammary epithelium and differentiates into both myoepithelial and luminal cells. The transplant assays might therefore uncover a regenerative potential of Lgr5cells that would be inhibited during MG pubertal development. However, in all the previous studies, depletion of a specific cell population in the presence of all the other cells was not attempted. We now have demonstrated that, in the MG, the unique approach of specific Lgr5cell depletion resulted in significantly impaired organogenesis, indicating that Lgr5cells are required during both regeneration from transplanted MECs but also, and more importantly, during physiological pubertal development.