The mammary gland is composed of multiple types of epithelial cells, which are generated by mammary stem cells (MaSCs) residing at the top of the hierarchy1,2. However, the existence of these multipotent MaSCs remains controversial and the nature of such cells is unknown3,4. Here we demonstrate that protein C receptor (Procr), a novel Wnt target in the mammary gland, marks a unique population of multipotent mouse MaSCs. Procr-positive cells localize to the basal layer, exhibit epithelial-to-mesenchymal transition characteristics, and express low levels of basal keratins. Procr-expressing cells have a high regenerative capacity in transplantation assays and differentiate into all lineages of the mammary epithelium by lineage tracing. These results define a novel multipotent mammary stem cell population that could be important in the initiation of breast cancer.
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The screening was initiated in the laboratory of R. Nusse and we are grateful for his support and generosity. We thank Y. Zhang and Biocytogen for assistance in knock-in mouse generation, and Q. Yin and H. Fang for technical support in RNA-seq analysis. We are grateful to C.-C. Hui and E. Verheyen for critical reading of the manuscript. We thank D. Li for helpful discussion. This work is supported by grants from the Ministry of Science and Technology of China (2014CB964800, 2012CB945000 to Y.A.Z., 2014CB910600 to L.Y.), National Natural Science Foundation of China (31171421, 31371500 to Y.A.Z., 31201098 to C.C.), the Chinese Academy of Sciences (XDA01010307, 2010OHTP03 to Y.A.Z., 2012OHTP08 to L.Y.), Shanghai Municipal Science and Technology Commission (12PJ1410100 to Y.A.Z.).
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Microarray of three-dimensional cultured basal cells in the presence of Wnt3A versus vehicle. 1 and 2 represent two independent experiments. See Methods for details. qPCR indicated that Procr is upregulated in basal cells cultured in the presence of Wnt3A compared with cells grown in the absence of Wnt3A. Data are pooled from three independent experiments. Data are presented as mean ± s.d. ***P < 0.01. c–g, The 4th inguinal mammary glands harvested from 2-week-old (c), 5-week-old (d), 8-week-old (e), pregnant day 14.5 (f) and 2 weeks post-weaning (g) CD1 mice were analysed by FACS. Procr+ cells were distributed in basal cells (ranging from 2.9% to 8.8%) and stromal fibroblasts (from 17.2% to 30.5%). No Procr+ cells were detected in luminal cells at any postnatal stage.
a, Basal (Lin− CD24+ CD29hi), Procr+ CD24+ CD29hi and Procr− CD24+ CD29hi cells were FACS-isolated and placed in Matrigel to assess the colony-formation ability. Data are pooled from four independent experiments. ***P < 0.01. b, Targeting strategy to generate the ProcrCreERT2-IRES-tdTomato/+ knock-in (KI) mouse. Designs of Southern blot probe and genotyping primers are as indicated. c, Southern blot analysis with a 5′ external probe of EcoRI-digested DNA from mouse embryonic stem cells, showing a 5.7 kb band in addition to the 7.7 kb wild-type (WT) band in clones that have undergone homologous recombination at the Procr locus. d, e, Embryos resulting from a cross of heterozygous male and female mice were dissected at E10.5 (d). Genotyping PCR indicated the proper distribution of wild type and heterozygotes as Mendel’s law of segregation, and that homozygotes were lethal before this time point as the embryo had mostly been absorbed (d, e). One of three similar experiments is shown.
Extended Data Figure 3 Procr+ cells and Lgr5+ cells are mutually exclusive populations in the mammary gland, while Procr+ cells and Axin2+ cells are largely non-overlapping in mammary basal cells.
a, Procr+ CD24+ CD29hi and Procr− CD24+ CD29hi cells were FACS-isolated and analysed by qPCR. Procr+ cells expressed significantly lower levels of Lgr5 compared with Procr− cells. Data are pooled from three independent experiments. ***P < 0.01. b, Cells isolated from Lgr5–GFP mammary gland were analysed for the expression of GFP and Procr. 5.8% of basal cells (Lin− CD24+ CD29hi) were Lgr5–GFP+ cells, while 2.4% of basal cells were Procr+ cells. These two populations were not overlapped. c, Three basal subpopulations as indicated were FACS-isolated and cultured in Matrigel for colony formation. Only Procr+ Lgr5−cells formed colonies whereas Procr− Lgr5+ and Procr− Lgr5− cells could not. Data are presented as mean ± s.d. Scale bars, 20 μm. ***P < 0.01. d, Recipient fat pads were injected with freshly sorted basal subpopulation cells as indicated and harvested at 8 weeks after surgery. Procr+ Lgr5− cells efficiently formed new mammary glands (frequency 1/14.4). Comparably, Procr− Lgr5+ cells had significantly lower reconstitution efficiency (1/165.4). Procr− Lgr5− cells were not able to reconstitute. e, Procr+CD24+ CD29hi and Procr− CD24+ CD29hi cells were FACS-isolated and analysed by qPCR. No significant difference in Axin2 level was detected in the two populations. Data are pooled from three independent experiments. ***P < 0.01. f, Procr+ CD24+ CD29hi and Procr− CD24+ CD29hi cells were isolated from Axin2-lacZ mammary gland and underwent X-gal staining. About 1.5% of Procr+ CD24+ CD29hi cells were Axin2-lacZ+, while 6.0% of Procr− CD24+ CD29hi cells were Axin2-lacZ+. Data are pooled from two independent experiments (n = 1,085 cells and n = 1,103 cells).
a–e, The 4th inguinal mammary glands harvested from E18.5 (a), P1.5 (b), 5-week-old (c, d) and 8-week-old (e) ProcrCreERT2-IRES-tdTomato/+ mice were analysed by whole-mount confocal imaging. Individual tdTomato+ cells were dispersedly located in all stages of mammary ducts. tdTomato+ cells were not found in TEBs of 5-week-old glands (c). A minimum of 50 TEBs and 50 ducts were analysed. Scale bars, 100 μm. f–i, Analysis of proliferative cells in TEBs and ducts of ProcrCreERT2-IRES-tdTomato mice at 3 h after EdU injection. Five-week-old TEBs exhibited abundant EdU+ cells (green) but no Procr+ cells (red) (f). EdU+ Procr+ cells (yellow) were found in both 5-week-old ducts (g) and 8-week-old mammary gland (h). Quantification is shown in i. Scale bars, 20 μm.
Extended Data Figure 5 Quantitative clonal analysis of Procr-labelled cells in mammary glands induced in puberty.
a–f, The number of basal and luminal cells in individual GFP+ clones were scored in ProcrCreERT2/+;R26mTmG/+ mammary glands after 3 weeks (a–c) or 6 weeks (d–f) induction. Basal cell numbers are shown along the y-axis, and luminal cell numbers are shown along the x-axis. Red shading indicates the relative frequency of certain clone composition, with deeper shading indicating higher frequency. d, Note that the deeper shading boxes shifted to the right in tracing experiments undertaken for a longer period. b, In clones after 3-week tracing, 72.4% were bi-lineage, 14.8% were solely basal cells derived from Procr+ cell division, 12.8% were single basal cells that had not divided. c, Among two-cell clones, 64.9% were composed of one basal cell and one luminal cell, while 35.1% consisted of two basal cells. d, e, In clones after 6-week tracing, the proportion of bi-lineage clone increased to 93%, while the percentage of the other two groups decreased to 3.9% and 3.1%. f, In two-cell clones, bi-lineage clones increased to 85.2%, while clones consisting of two basal cells decreased to 4.8%. n = 4 mice for 3-week tracing and n = 3 mice for 6-week tracing.
Extended Data Figure 6 Procr+ cells are long-lived multipotent MaSCs retained beyond multiple rounds of pregnancy.
a–d, Tamoxifen (TAM) was administered in 5-week-old ProcrCreERT2/+;R26mTmG/+ mice. Labelled cell contribution was analysed as illustrated in a. b–e, FACS analysis indicating that GFP+ cells are distributed in both basal and luminal layer in mid-2nd pregnancy (b, c) and mid-3rd pregnancy (d, e). f, Quantification of GFP+ cells indicating no difference in the percentage of GFP+ basal cells between nulliparous mice and multiparous mice that have gone through three complete cycles of pregnancy and involution. n = 3 mice. c, e, f, Data are presented as mean ± s.d.
a–g,Tamoxifen (TAM) was administered in 8-week old ProcrCreERT2/+;R26mTmG/+ mice. Labelled cell contribution was analysed after 3-week or 6-week induction. After 3 weeks, FACS analysis indicated that GFP+ cells were distributed in both basal and luminal layers (b, c). Immunostaining in sections showed the clonal expansion of GFP+ cells and confirmed their distribution in both basal and luminal layers. Basal cells were marked by K14, while cells apical to K14+ cells were luminal cells (arrow and arrowhead in d). Clonal analysis indicated that bi-lineage clones are the majority in all clones (74.2%) (e, f), and in two-cell clones (70.2%) (g). h–j, Clonal analysis of 6-week induction indicating that clone sizes are larger (red shaded boxes shifted to the right) (h), bi-lineage clone percentage has also increased to 94% in all clones (i) and to 89.5% in two-cell clones (j). k–n, Mammary glands were analysed at pregnant day 14.5 after tamoxifen administration at 8 weeks. GFP+ cells were in both basal and luminal layers as indicated by FACS analysis (l, m). GFP+ cells contributed to alveologenesis by immunohistochemistry analysis in sections (n). n = 3 mice for 3-week tracing and n = 3 mice for 6-week tracing. Scale bars, 20 μm. c, m, Data are presented as mean ± s.d.
a–g, Tamoxifen (TAM) was administered in pregnant day 18.5 mothers bearing ProcrCreERT2/+;R26mTmG/+ mice. Labelled cell contribution in the pups was analysed after 8-week induction (a). FACS analysis indicated that GFP+ cells are distributed in both basal and luminal layers (b, c). Immunostaining in sections showed the clonal expansion of GFP+ cells and confirmed their distribution in both basal and luminal layers (arrow and arrowhead in d). Scale bar, 20 μm. Clonal analysis indicated that bi-lineage clones are the majority in all clones (97.7%) (e, f), and in two-cell clones (97.1%) (g). n = 5 mice. c, Data are presented as mean ± s.d. h–n, Tamoxifen was administered in P0.5 ProcrCreERT2/+;R26mTmG/+ mice. Labelled cell contribution was analysed after 8-week induction (h). FACS analysis indicated that GFP+ cells are distributed in both basal and luminal layers (i, j). Immunostaining in sections showed the clonal expansion of GFP+ cells and confirmed their distribution in both basal and luminal layers. Basal cells were marked by K14, while cells apical to K14+ cells were luminal cells (arrows and arrowhead in k). Scale bar, 20 μm. Clonal analysis indicated that bi-lineage clones are the majority in all clones (98.5%) (l, m), and in two-cell clones (94.4%) (n). n = 5 mice. j, Data are presented as mean ± s.d.
a–d, Tamoxifen (TAM) was administered in 2-week old ProcrCreERT2/+;R26mTmG/+ mice. Labelled cell contribution was analysed at 8 weeks. FACS analysis indicated that GFP+ cells are distributed in both basal and luminal layers (b, c). Immunostaining in sections showed the clonal expansion of GFP+ cells and confirmed their distribution in both basal and luminal layers. Basal cells were marked by K14, while cells apical to K14+ cells were luminal cells (arrow and arrowhead in d). e–g, Clonal analysis indicated that bi-lineage clones are the majority in all clones (89.6%) (e, f), and in two-cell clones (78.8%) (g). n = 4 mice. h–k, Mammary glands were analysed at day 14.5 gestation after tamoxifen administration at 2 weeks. GFP+ cells were in both basal and luminal layers as indicated by FACS analysis (i, j). GFP+ cells contributed to alveologenesis by immunohistochemistry analysis in sections (k). Scale bars, 20 μm. c, j, Data are presented as mean ± s.d.
a, Schematic illustration of targeted ablation of Procr+ cells using the Procr-CreERT2 model to drive expression of DTA. b, Tamoxifen (TAM) was administered every 3 days a total of three times followed by analysing the 4th mammary gland. c–e, Whole-mount imaging of the mammary epithelium at P42. The lymph node (L.N.) is indicated. Both the oil-treated control mammary epithelium (c) and the tamoxifen-treated R26DTA/+ control mammary epithelium (d) had grown to the distal edge of the fat pad. Tamoxifen administration in ProcrCreERT2/+;R26DTA/+ mice largely prevented the growth of the epithelium: the forefront of the epithelium halted at a position close to where the forefront was at the initiation of cell ablation (slightly past the lymph node) (e). Scale bars, 2 mm. f, Quantification of the distance from the epithelium forefront to the lymph node indicated that epithelium extension in the Procr+ cell-ablation group is largely compromised (comparing e with d or c). ***P < 0.01. NS, not significant. g, h, FACS analysis indicating that Procr+ basal and stromal cells are ablated (fourfold and twofold). n = 3 mice. The role of Procr+ stromal cells in this study should also be taken into consideration as they were also affected by the ablation. Nonetheless, the reduction of Procr+ stromal cells was not as pronounced as Procr+ basal cells, probably due to the less proliferative nature of Procr+ fibroblasts, thereby fewer progeny cells were affected. Data are presented as mean ± s.d. ***P < 0.01. i, Multipotent and unipotent MaSCs coexist in the mammary epithelial cell hierarchy. Multipotent MaSCs are characterized by Lin− CD24+ CD29hi Procr+ K5low K14low, and express EMT features. Multipotent MaSCs generate all differentiated cell types, as determined by lineage tracing, and display the highest repopulation efficiency by transplantation. Basal-committed MaSCs are destined for basal cells in development, yet can repopulate both basal and luminal cells in transplantation, underlining the plasticity of basal-committed MaSCs in response to intervention. Luminal progenitors contribute to only luminal cells in lineage tracing and are not able to repopulate in transplantation. Their markers are as previously reported3,4,27.
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Wang, D., Cai, C., Dong, X. et al. Identification of multipotent mammary stem cells by protein C receptor expression. Nature 517, 81–84 (2015). https://doi.org/10.1038/nature13851
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