Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium

All experiments were conducted in genetically homogenous FVB/NHanHsd mice purchased from Harlan Laboratories. The mice were bred and maintained in the animal facility of the Friedrich Miescher Institute, according to the Swiss guidelines on animal experimentation. All experiments were performed under permit 2159-2, in accordance with the animal-welfare ordinance and approved by the cantonal veterinary office of Basel Stadt, Switzerland. For the early-pregnancy protocol, mice were time-mated when 42 days old and allowed to lactate for 21 days. The postweaning period until cell harvest was 40 days, unless stated otherwise. To control for the estrus cycle in the transcriptome analyses, at least five mice were grouped for gland harvesting. For immunohistochemical analyses and determination of blood progesterone levels, mice in estrus were used, as assessed by the presence of a vaginal plug after an overnight mating. Age-matched virgin control mice were maintained under the same conditions as parous mice.

Whole mounts were prepared by fixing the glands on glass slides with methacarnoy solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 4 hours at room temperature. The mounts were hydrated by sequential incubation in ethanol solutions of decreasing concentration: 100%, (overnight), 70%, 50%, and 30% (15 minutes each), distilled water (2 × 5 minutes), and stained overnight with an aqueous solution of 2% carmine (Sigma, Buchs, Switzerland) and 5% aluminum potassium sulfate (Sigma, Buchs, Switzerland). The mounts were dehydrated in ethanol solutions (70%, 90%, 95%, and 2 × 100%, for 15 minutes each) and cleared with xylene overnight. Images were captured with an Epson Expression 1600 Pro scanner.

The fourth mammary glands from virgin and parous mice were collected after lymph node removal and pooled. Mammary epithelial organoids were prepared as described [26]. Adipocytes were removed by repeated centrifugations (300 g). Red blood cells (RBCs) were eliminated by incubation with 8.3 g/L ammonium chloride (pH 7.5) for 5 minutes. The number of fibroblasts in gland extracts was reduced by their attachment to polystyrene cell-culture flasks (Corning, Buchs, Switzerland) during a 45-minute incubation step in Dulbecco modified Eagle medium (Invitrogen, Zug, Switzerland) with 10% FCS (Sigma, Buchs, Switzerland) at 37°C/5% CO₂ [26]. The epithelial organoids were directly processed to single-cell suspensions by digestion in Hyclone HyQTase (ThermoScientific, Lausanne, Switzerland) with gentle pipetting for 15 minutes at 37°C. The cell suspension was filtered through a 40-μm cell strainer (BD Falcon, Basel, Switzerland), and the final cell suspension pelleted at 650 g for 4 minutes.

Cells were labeled as previously described [26] by using the antibodies PE-Cy7-CD45 (1:33), FITC-CD24 (1:40), PE-CD49f (1:40), and APC-Sca1 (1:40). Detailed antibody information is given later. DAPI (0.2%, Invitrogen, Zug, Switzerland) was added 10 minutes before cell sorting (1:250). FACS was carried out on a MoFlo cell sorter (Becton Dickinson, Basel, Switzerland). Cells were gated based on their forward- and side-scatter profiles (FS Area and SS Area). A time-of-flight approach (pulse width) was used to exclude doublets and higher-order cell clumps. Dead cells (DAPI bright) and immune cells (CD45⁺) were gated out (see Additional file 1). The gate for basal stem/progenitor cells was set at the top 5% of CD49f-expressing cells, as described [19, 21] (see Figure 1). Routine examination of the sorted mammary epithelial cell subpopulations showed a degree of purification higher than 95%.

Freshly sorted cells of each subpopulation (500 cells) were plated onto irradiated 3T3L1 feeder cells (5,000 cells) in 24-well Primaria plates (Becton Dickinson, Basel, Switzerland). The cells were cultured in DMEM/Ham F12 (Invitrogen, Zug, Switzerland) with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin (Invitrogen, Zug, Switzerland), 5 μg/ml bovine pancreatic insulin (Sigma, cell culture-tested solution, Buchs, Switzerland), 10 ng/ml mouse EGF (BD Biosciences, Basel, Switzerland), and 10 ng/ml cholera toxin (Sigma, Buchs, Switzerland). After 24 hours, the medium was renewed, and 4 days later, the colonies were fixed with acetone/methanol (1:1), washed, and rehydrated with PBS and 0.05% NaN₃. In separate rescue experiments with selected mammary epithelial cells from parous mice, the incubation medium was supplemented with recombinant mouse Wnt4 (R&D Systems, Abingdon, UK) at 500 ng/ml. For quantification, colonies were immunofluorescently stained with Krt18 and Krt14 antibodies and with Hoechst 33342. The stained colonies were imaged with a MacroFluo Z16 microscope (Leica, Heerbrugg, Switzerland) at 2× magnification, and a Z1 microscope (Zeiss, Feldbach, Switzerland) at 5× magnification. The feeder cells served as negative controls for Krt18 and Krt14 staining. The number of colonies (that is, clusters of more than three cells [23]) per well was determined manually. Colonies defined as Krt18/Krt14 double-positive were double positive over a minimum of 20% of colony area.

Freshly sorted basal stem/progenitor cells were resuspended in PBS with 50% Matrigel (BD Biosciences, Basel, Switzerland) and injected (30 μl) in limiting dilution numbers into the cleared fourth mammary glands of 3-week-old syngeneic female mice. Glands from recipient mice were harvested 8 to 9 weeks after transplantation, processed as whole mounts (see earlier), and scanned with an Epson Expression 1600 Pro scanner. The number of outgrowths was counted, with an outgrowth defined as an epithelial structure with ducts starting from a central point and with lobules and/or terminal end buds (see Additional file 2) [27].

Microarray analyses were performed on unsorted total mammary cell suspensions (see Figure 2) and on FACS-sorted mammary cell subpopulations (see Figures 3 and 4, as well as Table 1). For unsorted mammary cells, RNA was extracted by using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Genomic DNA was removed by using gDNA Eliminator Mini Spin Columns (Qiagen, Hilden, Germany). The RNA concentration was measured with a Nanodrop 1000 machine, and RNA quality determined with an Agilent 2100 bioanalyzer and RNA Nano Chips. Aliquots of 100 ng of isolated total RNA were amplified by using the Ambion WT Expression kit (Ambion, Zug, Switzerland). For FACS-sorted mammary epithelial cell subpopulations, total RNA was isolated by using the Arcturus PicoPure Frozen RNA Isolation Kit (Life Technologies, Zug, Switzerland). Genomic DNA was removed by using RNase-Free DNase (Qiagen, Hilden, Germany), the RNA concentration determined by using the RiboGreen Assay, and RNA quality assessed by using an Agilent 2100 bioanalyzer and RNA Pico Chips. Total RNA was used as the input for synthesis of amplified cDNA with the NuGen Ovation Pico WTA System (NuGen Inc., Leek, The Netherlands).

Resulting double-stranded cDNA was fragmented and labeled by using the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, High Wycombe, UK). Affymetrix Gene Chip Mouse gene 1.0 ST microarrays were hybridized according to the GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix, High Wycombe, UK) with a hybridization time of 16 hours. The Affymetrix Fluidics protocol FS450_0007 was used for washing. Scanning was performed with Affymetrix GCC Scan Control Software v. 3.0.0.1214 on a GeneChip Scanner 3000 7 G with autoloader. Arrays were normalized, and probeset-level expression values calculated with R/Bioconductor's (v2.14) "affy" package by using the rma() function. Differential gene expression between experimental and control samples was determined by using linear modeling as implemented in the R/Bioconductor package "limma." For general analysis of gene expression in total mammary cell suspensions and in FACS-sorted mammary cell subpopulations, we used the cut-off linear fold change > 1.5, adjusted P value < 0.05, and average linear expression between conditions greater than 4. To determine the 10 most up- or downregulated genes in FACS-sorted mammary epithelial cell subpopulations, a cut-off of linear fold change > 2.0, adjusted P value < 0.001, and average linear expression between conditions ave > 4 was used. For FACS-sorted mammary epithelial cell subpopulations both resulting lists of differential genes were imported into Ingenuity IPA (Ingenuity, content version 12710793) for pathway analysis. Gene-set enrichments in FACS-sorted mammary epithelial cell subpopulations were determined by using the JAVA application from the Broad Institute v2.0 [28, 29] and gene sets v2.5 and v3.0, as well as custom gene sets (see Additional file 3).

The microarray data from this publication have been submitted to the NCBI Gene Expression Omnibus [30] and are deposited as GSE40875 (mouse mammary cell subtypes), GSE40876 (total mammary epithelial cells in mice), and GSE40877 (both total mammary epithelial cells and mammary cell subtypes in mice).

RNA was isolated as described earlier and converted to cDNA by using the WT-Ovation Exon Module Version 1.0 (NuGen Inc., Leek, The Netherlands). Real-time PCR was performed on the unamplified cDNA corresponding to the specified number of cells or on 25 ng of amplified cDNA (by using the NuGen Ovation Pico WTA System (NuGen Inc., Leek, The Netherlands) for amplification). The TaqMan probe-based system was applied in combination with the TaqMan Universal PCR Master Mix (Applied Biosystems, Zug, Switzerland). The probe IDs are given later. Cycling was performed with 7500 Fast and Step OnePlus Real-Time PCR Systems (both from Applied Biosystems, Zug, Switzerland).

These were the probes: Areg (Mm00437583_m1), Axin2 (Mm00443610_m1), B2M (Mm00437762_m1), Cdk8 (Mm01223097_m1), Cel (Mm00486975_m1), Csn1s1 (Mm00514430_m1), Csn2 (Mm04207885_m1), Dsc2 (Mm00516355_m1), Dusp1 (Mm00457274_g1), Esr1 (Mm00433149_m1), Fst (Mm00514982_m1), Gata3 (Mm00484683_m1), Hprt (Mm00446968_m1), Id3 (Mm00492575_m1), Igfbp3 (Mm_01187817_m1), Jag2 (Mm01325629_m1), Krt8 (Mm00835759_m1), Krt14 (Mm00516876_m1), Krt15 (Mm00492972_m1), Krt19 (Mm00492980_m1), Lgr5 (Mm00438890_m1), Ltf (Mm00434787_m1), Maml2 (Mm00620617_m1), Myc (Mm00487804_m1), Pgr (Mm00435628_m1), Sfrp1 (Mm00489161_m1), Ubc (Mm01201237_m1), Vcan (Mm01283063_m1), Wnt4 (Mm01194003_m1).

For single-cell staining, freshly sorted cells were allowed to air-dry on poly-L-lysine-coated slides and stored at -20°C. The dried cells were blocked with PBS, 2.5% goat serum, and 0.05% NaN₃ for 60 minutes under UV light. The UV treatment was used to attenuate residual fluorescence from bound FACS antibodies. Primary antibody staining was performed overnight at 4°C by using Krt18 antibody (1:1,000) and Krt14 antibody (1:500) as luminal and basal cell markers, respectively. Secondary antibody staining was carried out for 60 minutes at room temperature by using anti-guinea pig Ig-Alexa488 (1:500) and anti-rabbit Ig-Alexa546 (1:1,000). Hoechst 33342 (0.5 μg/ml; Invitrogen, Zug, Switzerland) staining was performed for 10 minutes at room temperature. The stained cells were mounted with ProLong Gold antifade reagent (Invitrogen, Zug, Switzerland) and imaged with a Z1 microscope (Zeiss, Feldbach, Switzerland) at 63× magnification. No primary antibody was added as a negative control.

The fourth mammary glands of parous or age-matched virgin control mice were collected 40 days after weaning from mice in estrus. The glands were fixed in 4% PFA and embedded in paraffin. For immunostaining with ERα and PR, the sections were dewaxed and subjected to antigen retrieval by boiling in 10 mM citrate buffer for 10 minutes. Subsequently, the sections were cooled to room temperature, quenched for 10 minutes with PBS and 3% H₂O₂, and blocked for 30 to 60 minutes with PBS and 2.5% NGS. ERα and PR primary antibody staining was performed overnight at 4°C at a 1:1,000 (ERα) and a 1:200 (PR) dilution. Secondary antibody staining was carried out for 30 to 60 minutes at room temperature with biotinylated anti-rabbit IgG. Immunohistochemistry for versican, β-catenin, and p21 was performed on the Ventana DiscoveryXT instrument (Roche Diagnostics, Rotkreuz, Switzerland) by using the Research IHC Dap Map XT procedure. In brief, dewaxing was performed in the machine, and slides were pretreated with mildCC1 (versican and p21) or standardCC1 (β-catenin) (Roche Diagnostics, Rotkreuz, Switzerland). Primary antibodies were incubated at 37°C for 1 hour at the following dilutions: versican, 1:50; p21, 1:50; and β-catenin, 1:500. As secondary antibody, Immpress, an anti-rabbit HRP conjugated polymer, was used. All slides were counterstained with Hematoxylin II (Roche Diagnostics, Rotkreuz, Switzerland) and Bluing Reagent (Roche Diagnostics, Rotkreuz, Switzerland). Images were captured at 20-fold (ERα or PR) or 40-fold (versican, β-catenin, p21) magnification by using an Eclipse E600 microscope (Nikon, Egg, Switzerland). For quantification of ERα and PR positivity, at least 2,000 epithelial cells per mouse were counted. For quantification of nuclear β-catenin and p21, at least 500 epithelial cells per mouse were counted. The quantification of versican was performed with the MATLAB software by using color segmentation based on Mahalanobis distance to determine the pixels with a particular RGB-color distribution [31].

For flow cytometry, the following anti-mouse antibodies were used: PE-Cy7-CD45 (clone 30-F11), FITC-CD24 (clone M1/69), PE-CD49f (clone GoH3) (all from BD Pharmingen, Basel, Switzerland), and APC-Sca1 (clone E13-161.7, from Biolegend, San Diego, USA).

For immunofluorescence staining, the primary anti-mouse antibodies used were guinea pig keratin 18 (Krt18; Fitzgerald; catalog no. GP11, North Acton, USA) and rabbit keratin 14 (Krt14; ThermoScientific, catalog no. RB-9020, Lausanne, Switzerland). The secondary antibodies used were anti-guinea pig Ig-Alexa488 and anti-rabbit Ig-Alexa546 (Molecular Probe, Invitrogen, Zug, Switzerland).

For immunohistochemistry staining, the primary antibodies included estrogen receptor alpha (ERα; Santa Cruz Biotechnology; catalog no. SC-542, Dallas, USA), progesterone receptor (PR; Clone SP2, ThermoScientific; catalog no. RM-9102, Lausanne, Switzerland), versican (Millipore/Chemicon; catalog no. ab1033, Billerica, USA), β-catenin (Cell Signaling; catalog no. 9587, Danvers, USA), and p21 (Abcam; catalog no. ab2961, Cambridge, UK). The secondary antibodies used were biotinylated anti-rabbit IgG (Vector Labs; catalog no. BA-1000, Petersborough, UK) and anti-rabbit HRP conjugated polymer Immpress (Vector Labs; catalog no. MP-7401, Petersborough, UK).

The limited dilution transplantation data were analyzed statistically as published previously [18]. The two-tailed unpaired Student t test was used to determine statistical significance of comparisons.

The MARA model [32] combines knowledge of gene-expression levels (measured by microarray) with transcription-factor binding sites to answer the question of which transcription factors are driving expression changes in mammary stem/progenitor cells in parous as compared with age-matched virgin control mice. Specifically, log-expression levels of all genes present on the microarray were modeled as linear combinations of transcription factor activities. The coefficients of these combinations were determined by the number of transcription-factor binding sites in the proximal promoter regions. For each transcription-factor binding motif m and each sample (microarray) s, we estimated the activity A _ms with the corresponding error. Furthermore, we quantified the significance of activity change of each binding motif in parous as compared with virgin control mice.

To investigate the influence of early parity on the proportions of mammary epithelial cell subtypes, we first established FACS profiles of epithelial cell subpopulations in virgin FVB control mice [21, 26]. Luminal CD24^+High Sca1⁺ cells, luminal CD24^+High Sca1^- cells, basal CD24^+Low Sca1^- CD49f^High cells, and basal CD24^+Low Sca1^- CD49f^Low myoepithelial cells were isolated from these mice (Figure 1A). Use of the established cell markers keratin 18 (Krt18) and keratin 14 (Krt14) confirmed the luminal and basal origin of the isolated cell subpopulations, and qPCR for CD49f and Sca1 affirmed the purity of the isolated cell subpopulations (see Additional file 4). Because basal CD49f^High cells are considered to be enriched for basal mammary stem/progenitor cells in virgin mice [19, 21], the term "basal CD49f^High cells" is used synonymously with "basal stem/progenitor cells" throughout this article. To extend these analyses to parous mice, we confirmed that involution was complete 28 and 40 days after weaning (see Additional file 5). To allow a margin of safety, 40 days after weaning was used for all subsequent cell-isolation experiments in a standardized parturition protocol with mating at 42 days (Figure 1B). The FACS profiles of epithelial cell subpopulations from parous mice and age-matched virgin control mice were similar (Figure 1C, D; Additional file 1) with the exception of luminal Sca1⁺ cells, which decreased by about 50% in parous mice (P = 0.02). Of note, luminal Sca1⁺ cells have been shown to be enriched for hormone receptor-positive cells [17]. This was verified by qPCR (see Additional file 6). These data demonstrate that the adopted experimental procedure permits the isolation of all epithelial cell subpopulations from parous mice, including basal stem/progenitor cells, at levels adequate for transcriptomic and functional analyses.

Next we investigated the effects of early pregnancy on the gene-expression profiles of the isolated mammary epithelial cell subpopulations. To control for the effect of the cell-isolation procedure on gene expression, we performed first a transcriptome and cluster analysis in non-FACS-sorted total mammary cell suspensions from age-matched virgin and parous mice (Figure 2A). The analysis showed that pregnancy induces an upregulation of many immunoglobulin and differentiation genes (for example, whey acidic protein Wap, and carboxyl ester lipase Cel) and a downregulation of growth factors (for example, amphiregulin Areg, betacellulin Btc, tumor-associated calcium signal transducer 2 Tacstd2), and extracellular matrix (ECM) elements (for example, laminin, gamma 2 Lamc2, desmocollin 2 Dsc2). These data are consistent with the published pregnancy-induced gene signature determined in snap-frozen rodent mammary glands [9, 10]. Thus, the gene signature was not lost during the isolation procedure, confirming the validity of our experimental system. Furthermore and most interestingly, a novel 3.4-fold downregulation of the Wnt signaling protein Wnt4 was observed in cells from parous mice (Figure 2). These microarray data were validated by qPCR for four genes, including Wnt4. Importantly, the expression of the luminal marker Krt8 was not altered on parity, demonstrating that the observed changes in gene expression were independent of unspecific alterations in total luminal cell numbers (Figure 2B).

Because the number of isolated basal stem/progenitor cells for gene-profiling analysis was limited compared with the other cell subpopulations (that is, myoepithelial, luminal Sca1^-, and luminal Sca1⁺ cells), it was important to evaluate the influence of different cell numbers on transcriptome analysis. As shown in Additional Material, we found that it is valid to use cell numbers in the range of 2,000 to 50,000 cells for comparison of transcriptomes from different mammary cell subpopulations (Additional file 6). Furthermore, qPCR analysis of nonamplified and amplified cDNA for known basal (Krt14) and luminal (Krt8; Krt19) cell marker genes confirmed that the amplification process of the microarray analysis was unbiased (Additional file 6).

In the subsequent transcriptome analysis of FACS-sorted mammary epithelial cell subpopulations from age-matched virgin control and parous mice, all mammary epithelial cell subpopulations, except immune cell-depleted stromal cells, showed parity-induced changes in gene expression with by far the most prominent effects being scored for basal stem/progenitor cells (Figure 3A). Furthermore, although differentiation genes were upregulated in all epithelial cell subpopulations, the strongest prodifferentiation effects were seen in basal stem/progenitor cells from parous mice. For example, casein alpha 1 (Csn1s1), casein beta (Csn2), and lactotransferrin (Ltf) were upregulated 41-, 19-, and 14-fold, respectively (Figure 3B). Apart from these differentiation genes, the Notch co-activator Maml2 was found among the 10 most-upregulated genes (by fold change and/or by P value) in basal stem/progenitor cells from parous mice. In contrast, the 10 most-downregulated genes included the Wnt target and co-receptor Lgr5, the Wnt target and epithelial stem cell marker keratin 15 (Krt15) [33], and the Wnt targets versican (Vcan) and Igfbp3 [34] (Figure 3B). Extension of the analysis to all data for signaling-pathway genes revealed further Wnt target genes, which were downregulated (for example, CD44 and follistatin (Fst) [35, 36]), and the Wnt inhibitor Sfrp1, which was upregulated in basal stem/progenitor cells from parous mice. Moreover, in the same epithelial cell subpopulation of parous mice, the Notch ligand Jag2 and the Notch target genes Gata3, Id3, and Dusp1 were upregulated, whereas the Notch inhibitor Itch and the Maml2 RBP-J complex inhibitors Cdk8 and Pak1 [37] were downregulated (Figure 3B). These gene-expression profiling data were validated with qPCR for several selected genes (Figure 3C). Furthermore, one of the classic Wnt target genes (versican) was examined on the protein level and found to be strongly downregulated in the basal compartment of mammary glands from parous mice (Figure 3D). Thus, the data demonstrate strong upregulation of differentiation genes, a downregulation of Wnt target genes, and an increase in Notch signaling in the basal stem/progenitor-cell subpopulation of parous mice.

Further verification of these conclusions came from Motif Activity Response Analysis (MARA) of transcription factor activities (see Methods) [38]. The canonical Wnt transcription factor LEF1/TCF7 was shown to have significantly decreased activity in basal stem/progenitor cells from parous mice (Figure 4A), which is consistent with the observed decrease in canonical Wnt signaling in the previous gene-expression profile analysis.

Final confirmation of parity-induced downregulation of Wnt signaling in mammary glands was provided by immunohistochemical staining of mammary gland sections from age-matched virgin control and parous mice for β-catenin. As illustrated in Figure 4B, nuclear β-catenin was observed in basal but not in luminal mammary epithelial cells of virgin mice. In parous mice, the proportion of basal mammary epithelial cells positive for nuclear β-catenin was significantly decreased (Figure 4C). Because nuclear β-catenin is necessary for Wnt target gene expression, this finding represents an additional verification of parity-induced downregulation of Wnt signaling (Figure 3).

To assess whether the observed decrease in canonical Wnt and increase in Notch signaling were specific for basal stem/progenitor cells, we next investigated the enrichment of Wnt/Notch signaling genes over all genes altered in the various FACS-sorted mammary epithelial cell subpopulations from parous as compared with virgin control mice. Such analysis showed that canonical Wnt signaling was significantly downregulated in basal stem/progenitor but not in luminal Sca1^- or luminal Sca1⁺ cells from parous mice (Table 1; see Additional file 7). In myoepithelial cells, a trend toward a decrease in Wnt signaling was not significant when applying a very stringent cut-off (see Table 1 legend) and was probably due to contamination of the myoepithelial cell subpopulation with basal stem and/or progenitor cells. Similarly, Notch signaling was found to be significantly upregulated in basal stem/progenitor cells but in no other mammary epithelial cell subpopulation (Table 1). Whereas downregulation of Wnt and upregulation of Notch signaling were specific for basal stem/progenitor cells, similar enrichment analyses for genes involved in other signaling pathways revealed that the previously reported upregulation of the p53-p21 pathway [39] occurred in all epithelial cell subpopulations tested (Additional file 7).

Further bioinformatics analysis of the data with different software [40] provided a second line of verification of the specific decrease in Wnt signaling in basal stem/progenitor cells after parity. Performing transcription-factor activity analyses based on target gene expression, we found TCF3, an inhibitor of the canonical Wnt signaling pathway [41], to be the transcription factor with the highest z-score (Z = 3.521) and the protooncogene MYC to have a very low z-score (Z = -4.108) in basal stem/progenitor cells but not in other mammary epithelial cell subpopulations of parous mice (Figure 4D, z-score defined as (x-μ)/sd and used as a measure for transcription factor activity based on the expression levels of target genes). These findings confirm the results obtained in the MARA analysis. Furthermore, inhibition of MYC leads to upregulation of Sfrp1, which in turn inhibits canonical Wnt signaling [42]. Upregulation of Sfrp1 was observed in the microarray analysis and validated with qPCR (Figure 3B, C), thus directly reflecting the expected effects of the bioinformatic predictions.

Downregulation of canonical Wnt signaling and MYC activity would be expected to decrease the propensity for cancer, and indeed, in an analysis of biofunctions, a marked and consistent decrease in cancer-associated functions, was observed for basal stem/progenitor cells (Figure 4E), but no other mammary epithelial cell subpopulation. This potential anticancer phenotype of basal stem/progenitor cells was underscored by gene-enrichment analyses on all available pathway gene sets (see Additional file 8), which showed a strong downregulation of proliferation- and tumorigenesis-associated gene sets.

Because decreased Wnt signaling and increased Notch signaling have been shown to decrease in vitro and in vivo proliferation of basal stem/progenitor cells [43, 44], we next assessed the in vitro colony-formation capacities of mammary epithelial cell subpopulations from parous and age-matched virgin mice (Figure 5A). In virgin control mice, luminal Sca1^- cells had the highest colony-formation capacity, with an average of 107 colonies per well (Figure 5B). This strong clonogenic potential suggests a pronounced progenitor identity of luminal Sca1^- cells and is consistent with previous observations in younger virgin mice [16, 17]. A high colony-formation capacity was also observed for the basal stem/progenitor cells of virgin mice (63 colonies per well) (Figure 5B), which is consistent with the notion that CD49f^High cells contain a high proportion of basal progenitor cells as well as putative mammary stem cells [45]. With the exception of luminal Sca1^- cells, the colony-formation capacities of all epithelial cell subpopulations were lower in parous mice than in age-matched virgins (Figure 5B). Thereby, by far the most pronounced difference was observed in basal stem/progenitor cells (Figure 5B). A substantial decrease in the colony-formation capacity was also seen for the myoepithelial cell subpopulation, which also contains basal progenitor cells. Of note, basal stem/progenitor cells from parous mice did not die but remained as quiescent single cells or divided only once during 5 days of culture (Figure 5C).

The reduced progenitor potential of basal mammary epithelial cell subtypes and especially of basal stem/progenitor cells from parous mice was further confirmed by phenotypic analysis of colonies by colony size (cell number) and immunophenotyping of luminal and basal markers Krt18 and Krt14, respectively. The largest difference in colony size was observed for the basal stem/progenitor cell subpopulation, where parity induced a decrease in colonies of ≥ 20 cells from 66% to 9% (Table 2A; Figure 5C). Considerably smaller parity-induced reductions in colony size were observed for the other epithelial cell subpopulations, although a substantial decrease was also seen for basal myoepithelial cells (Table 2A). With regard to Krt18/Krt14 double positivity, parity induced a reduction in Krt18/Krt14 double-positive colonies derived from basal stem/progenitor cells from 26% to 0 (Table 2A). In contrast, although Krt18/Krt14 double-positivity decreased by 27%, 57% of colonies derived from luminal Sca1^- cells of parous mice maintained double positivity (Table 2A). No effects of parity on Krt18/Krt14 double positivity were observed for colonies derived from luminal Sca1⁺ or basal myoepithelial cells (Table 2A), whereby myoepithelial cells did not give rise to double-positive colonies even when originating from virgin control mice. Thus, consistent with the basal mammary stem/progenitor cell subpopulation-specific reduction in the Wnt/Notch signaling ratio, parity decreased colony-formation capacity, in vitro proliferation potential (colony number and size), and Krt18/Krt14 double positivity most prominently in the basal stem/progenitor cell subpopulation.

To test their proliferation potential in vivo, we transplanted basal stem/progenitor cells into deepithelialized mammary glands ("cleared fat pads") [12]. It already was demonstrated that basal CD49f^High cells have the highest mammary gland reconstitution ability of all mammary cell subpopulations in virgin mice [16, 19]. Transplantation of FACS-isolated basal stem/progenitor cells into cleared fat pads demonstrated a significant decrease in the number of large outgrowths (≥ 10% and ≥ 25% of fat pad filled), indicating a decrease in the in vivo proliferation potential. Interestingly, when assessing also for rudimentary outgrowths (≥ 3% of fat pad filled), no significant difference in the number of outgrowths was observed between parous and virgin donors (Table 2B). Apart from the change in size, no qualitative differences were apparent between outgrowths from virgin and parous donors. In both cases, ductal as well as lobular structures were formed. Hence, early parity led to a reduction in in vivo mammary repopulating efficiency of FACS-isolated basal stem/progenitor cells, whereas their ability to differentiate into different mammary epithelial structures was maintained.

Having observed most of the expected functional consequences of a decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells from parous mice, we finally examined the possible cause of parity-induced reduction in canonical Wnt signaling and proliferation capacity in basal stem/progenitor cells. Because parity induced a greater than threefold decrease in Wnt ligand Wnt4 gene expression (Figure 2A, B), and Wnt4 is known to be secreted in response to progesterone by hormone-sensing luminal cells [46], thus inducing canonical Wnt signaling in mammary stem/progenitor cells [47], a parity-induced decrease in estrogen/progesterone-sensitive luminal cells could explain the overall decrease in Wnt signaling in mammary stem cells. This hypothesis is supported by the reduction in the proportion of luminal Sca1⁺ cells isolated from parous mice (Figure 1D) and by the demonstration that luminal Sca1⁺ cells are hormone receptor positive (Additional file 6) [17]. Furthermore, immunohistochemical analysis of mammary gland sections for estrogen receptor alpha (ERα) and its target progesterone receptor (PR) showed a twofold decrease in ERα- and a threefold decrease in PR-positive cells in parous compared with age-matched virgin control mice (Figure 6A, B). These results were additionally verified by qPCR in total mammary cell suspensions (Figure 6C). Notably, expression of the luminal marker Krt8 was similar in cell suspensions from parous and age-matched virgin control mice, supporting the conclusion of a specific decrease in hormone receptor-positive cells rather than a general cell loss after pregnancy. Furthermore, parity-induced reduction in progesterone-stimulated Wnt4 expression was independent of blood progesterone, because average blood progesterone concentrations were similar in parous mice and age-matched virgin control mice in estrus (see Additional file 9). Finally, supplementation of the culture medium with recombinant Wnt4 stimulated in vitro proliferation capacity of basal myoepithelial and basal stem/progenitor cells from parous mice by +138% ± 22% and +140% ± 17%, respectively (Figure 7A). In contrast, no significant effects of recombinant Wnt4 on the colony-formation capacity of luminal Sca1^- (+3.7% ± 3.0%) and luminal Sca1⁺ cells from parous mice (-5.1% ± 2.9%) were observed. These data strongly suggest a causal relation between reduced number of luminal progesterone receptor-positive/Wnt4-secreting cells and decreased Wnt/Notch signaling and proliferation potential of basal stem/progenitor cells after early pregnancy (Figure 7B).