Mammary stem cells have myoepithelial cell properties

Abstract

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin–myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

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Figure 1: Most mouse basal cells express αSMA.
Figure 2: Short-term culture increases MRU numbers by ~460-fold.
Figure 3: A high proportion of single-cell-derived basal colonies contain a MRU.
Figure 4: Myoepithelial cells have MRU activity.
Figure 5: Myoepithelial-cell-derived clones can undergo expansion within the basal layer of intact mammary glands and survive after multiple pregnancies.
Figure 6: The progeny of myoepithelial cells is restricted to the basal cell layer and survives after multiple pregnancies.

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Change history

  • 12 September 2014

    In the version of this Article published online, the authors omitted a key Agence Nationale de la Recherche funding grant (ANR-13-BSV2-0001) to Marina A. Glukhova from Acknowledgements. This error has now been corrected in all versions of the article.

References

  1. 1

    Franke, W. W. et al. Intermediate-sized filaments of the prekeratin type in myoepithelial cells. J. Cell Biol. 84, 633–654 (1980).

  2. 2

    Lazard, D. et al. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc. Natl Acad. Sci. USA 90, 999–1003 (1993).

  3. 3

    Chepko, G. et al. Differential alteration of stem and other cell populations in ducts and lobules of TGF[α] and c-Myc transgenic mouse mammary epithelium. Tissue Cell 37, 393–412 (2005).

  4. 4

    Smith, G. H. & Medina, D. A morphologically distinct candidate for an epithelial stem cell in mouse mammary gland. J. Cell Sci. 90, 173–183 (1988).

  5. 5

    Stingl, J. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006).

  6. 6

    Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006).

  7. 7

    Sleeman, K. E. et al. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J. Cell Biol. 176, 19–26 (2007).

  8. 8

    van Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189–193 (2011).

  9. 9

    van Amerongen, R., Bowman, A. N. & Nusse, R. Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11, 387–400 (2012).

  10. 10

    Taddei, I. et al. β1 Integrin deletion from the basal compartment of the mammary epithelium affects stem cells. Nat. Cell Biol. 10, 716–722 (2008).

  11. 11

    Rios, A. C., Fu, N. Y., Lindeman, G. J. & Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014).

  12. 12

    Shehata, M. et al. Phenotypic and functional characterization of the luminal cell hierarchy of the mammary gland. Breast Cancer Res. 14, R134 (2012).

  13. 13

    Gottlieb, C., Raju, U. & Greenwald, K. A. Myoepithelial cells in the differential diagnosis of complex benign and malignant breast lesions: an immunohistochemical study. Mod. Pathol. 3, 135–140 (1990).

  14. 14

    Wu, Y-J. et al. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell 31, 693–703 (1982).

  15. 15

    Inman, G. J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

  16. 16

    Flanagan, M. D. & Lin, S. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J. Biol. Chem. 255, 835–838 (1980).

  17. 17

    Spector, I., Shochet, N., Kashman, Y. & Groweiss, A. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219, 493–495 (1983).

  18. 18

    Bubb, M. R., Senderowicz, A. M., Sausville, E. A., Duncan, K. L. & Korn, E. D. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J. Biol. Chem. 269, 14869–14871 (1994).

  19. 19

    Ikenoya, M. et al. Inhibition of Rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. J. Neurochem. 81, 9–16 (2002).

  20. 20

    Chen, G., Hou, Z., Gulbranson, D. R. & Thomson, J. A. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240–248 (2010).

  21. 21

    Ohgushi, M. et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7, 225–239 (2010).

  22. 22

    Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 299, 1743–1747 (2003).

  23. 23

    Haaksma, C. J., Schwartz, R. J. & Tomasek, J. J. Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin. Biol. Reprod. 85, 13–21 (2011).

  24. 24

    Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

  25. 25

    Chepko, G. & Smith, G. H. Three division-competent, structurally-distinct cell populations contribute to murine mammary epithelial renewal. Tissue Cell 29, 239–253 (1997).

  26. 26

    Joshi, P. A. et al. Progesterone induces adult mammary stem cell expansion. Nature 465, 803–807 (2010).

  27. 27

    Yokota, T. et al. Bone marrow lacks a transplantable progenitor for smooth muscle type α-actin-expressing cells. Stem Cells 24, 13–22 (2006).

  28. 28

    Wendling, O., Bornert, J. M., Chambon, P. & Metzger, D. Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse. Genesis 47, 14–18 (2009).

  29. 29

    Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).

  30. 30

    Young, L. J. T. in Methods in Mammary Gland Biology and Breast Cancer Research (eds Ip, M. M. & Asch, B. B.) 67–74 (Kluwer/Plenum, 2000).

  31. 31

    Gentleman, R. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

  32. 32

    Cairns, J. M., Dunning, M. J., Ritchie, M. E., Russell, R. & Lynch, A. G. BASH: a tool for managing BeadArray spatial artefacts. Bioinformatics 24, 2921–2922 (2008).

  33. 33

    Dunning, M. J., Smith, M. L., Ritchie, M. E. & Tavaré, S. beadarray: R classes and methods for Illumina bead-based data. Bioinformatics 23, 2183–2184 (2007).

  34. 34

    Smyth, G. K. in Bioinformatics and Computational Biology Solutions using R and Bioconductor (eds Gentleman, R.et al.) 397–420 (Springer, 2005).

  35. 35

    Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009).

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Acknowledgements

We thank A. Saadi for contributing to data interpretation and A. Chiche, G. Carita, T. Makdessi and A. Di-Cicco for help with lineage-tracing experiments. We are grateful to P. Chambon and P. Soriano for providing mice. We thank M. Leeson and L. Tauzin for assistance with cell sorting; J. Atkinson and J. Miller for sectioning and immunohistochemistry; M. Osborne for conducting the microarrays; J. Gray for help with western blotting; S. Fre and V. Rodilla for advice on the cell sorting of R26mTmG mouse mammary cells. We thank F. Watt and S. Broad for useful discussions and providing FAD media. We thank S. Nourshargh, M. Finsterbusch and J. Brown for access to mammary tissue from transgenic mice. This work was funded by Cancer Research UK, Breast Cancer Campaign, the University of Cambridge, Hutchison Whampoa Limited, La Ligue Nationale Contre le Cancer (Equipe Labelisée 2013) and grants from Agence Nationale de la Recherche ANR-08-BLAN-0078-01 and ANR-13-BSV2-0001 to M.A.G.

Author information

M.D.P. conducted the experiments and co-wrote the manuscript. V.P., I.A.R., I.K., N.R., R.R.G. and M.S. conducted experiments. M.M.F. and M-A.D. conducted experiments, analysed data and contributed to data interpretation. M.F.O. provided mice and designed experiments. D.M. provided mice. S.M. analysed the microarray data. R.S. helped design flow cytometry experiments. M.A.G. and J.S. designed experiments, analysed data and co-wrote the manuscript.

Correspondence to Marina A. Glukhova or John Stingl.

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Competing interests

J. Stingl is a paid consultant for StemCell Technologies Inc.

Integrated supplementary information

Supplementary Figure 1 Flow cytometry gating strategy.

(a) Gating strategy to discriminate basal cells. (b) Comparison of the colony forming efficiency of sorted live cells exposed to DAPI and antibodies compared to unsorted live cells. Data is presented a the mean (±s.e.m.) of 6 independent experiments; P < 0.05 by 2-tailed Student’s t-test.

Supplementary Figure 2 Flow cytometry gating strategy to identify S/G2/M basal cells.

Supplementary Figure 3 Basal cells in S/G2/M cell cycle phases have higher MRU frequency and colony forming efficiency than basal cells in G0/G1 phases.

(a) Frequency of S/G2/M cells in basal EpCAMhigh and EpCAMlow cells as determined by Hoechst 33342 staining. Mean (±s.e.m.) of 4 independent experiments. P = 0.02 for G0/G1 and P = 0.03 for G2/M. (b) Distribution of G2/M cells within the basal population. Mean (±s.e.m.) of 4 independent experiments. (c) Images of basal cells in S/G2/M phases (Hoechst4n) and basal cells in G0/G1 phases (Hoechst2n) from the ImageStream imaging flow cytometer. One sample analysed. Scale bar = 10 μm. (d) MRU frequency and engraftment rates in basalHoechst4n and basalHoechst2n cells. Data pooled from 2 independent experiments. MRU distribution calculated by multiplying MRU frequency by the total number of cells for each population per gland and calculating the ratio of total MRU numbers between the 2 cell populations. (e) Colony forming efficiency of G0/G1 and S/G2/M basal cells. Mean (±s.e.m.) of 4 independent experiments. P = 0.005.

Supplementary Figure 4 Cytoskeletal remodelling and inhibition of TGFβ significantly influence basal colony formation.

(a) GeneGo pathway maps listed in order of significance; P < 10−15 P < 10−10 P < 10−5. (b) Basal cell CFE after addition of TGFβ1 at 200 pM and the TGFβ inhibitor SB 431542 hydrate (SB) at 10 μM to FAD media; mean (±s.e.m.) of 3 independent experiments for all conditions, except for when TGFβ and SB 431542 are tested individually, then the data is derived from 5 independent experiments. P = 0.02 for no treatment versus TGFβ, P = 0.04 for TGFβ versus SB + TGFβ, P = 0.02 for Y versus Y + TGFβ, P = 0.02 for Y + TGFβ versus Y + SB, P = 0.02 for Y + TGFβ versus Y + SB + TGFβ. (c) Effect of the actin modulators: latrunculin B, cytochalasin D and jasplakinolide on basal cell CFE. Data is presented as the mean (±s.e.m.). The number of independent experiments performed for each experimental condition is indicated by the number on top of the bars in the chart. Significant difference to no treatment control (P = 0.03 for 1 μM latrunculin B, P = 0.02 for 5 μM latrunculin B, P = 0.02 for 50 nM cytochalasin D, P = 0.01 for 100 nM cytochalasin D). † Significant difference to + Y treatment (P = 0.003 for 250 nM cytochalasin D, P = 0.02 for 25 nM jasplakinolide, P = 0.002 for 50 nM jasplakinolide). (d) The effect of the Rho kinase inhibitors Y-27632 (Y) and H1152 (H) and the myosin II inhibitor Blebbistatin (Bleb) on basal cell CFE. Data is presented as the mean (±s.e.m.). The number of independent experiments performed for each experimental condition is indicated by the number on top of the bars in the chart. Significant difference to no treatment (P = 0.007 for 1 μM Y, P = 0.00003 for 10 μM Y, P = 0.007 for 100 nM H, P = 0.0006 for 1 μM H, P = 0.0008 for 5 μM Bleb, P = 0.002 for 10 μM Bleb, P = 0.0001 for Y + Bleb).

Supplementary Figure 5 A high proportion of basal αSMA cells express the basal cytokeratins 5/14. Engraftments derived from myoepithelial cells can form secondary engraftments.

(a) Flow cytometry plot showing GFP expression (activity of the Acta2 promoter) in basal and luminal cells. (b) Flow cytometry plot showing GFP expression in basal EpCAMhigh and EpCAMlow cells. (c) Flow cytometry plot showing EpCAM expression in stromal, basal GFP, basal GFP+ and luminal cells. Three independent samples were analysed for panels ac. (d) Immunofluorescence images of flow-sorted basal αSMA andαSMA+ cells stained for CK5 and CK14. Isotype control inset. Scale bar = 100 μm. Mean (±s.e.m.) of 2 independent experiments. (e) Limiting dilution analysis of dissociated primary engraftments derived from αSMA+ basal cells. (f) Number of secondary engraftments derived from 1 mm3 fragments of primary outgrowths produced by Myh11+ basal cells and total basal cells. Data pooled from 2 independent experiments.

Supplementary Figure 6 The progeny of myoepithelial cells is restricted to the basal cell layer and survives after multiple pregnancies.

(a) Cytospots of sorted basal and luminal cells from Acta2-Cre-ERT2;Rosa26LacZ mice x-gal stained in suspension. Arrows point to LacZ-positive cells. Sorted cells from five independent samples were analysed. Scale bars = 10 μm. (b) Flow cytometry dot plots showing GFP expression in luminal and basal cell populations isolated from 13-day-pregnant Acta2-Cre-ERT2;R26mTmG mouse mammary glands. Experimental schedule was same as shown in Fig. 5e. Red ovals indicate luminal (L) and basal (B) cell populations. (c) A fragment of whole-mount x-gal stained mammary gland from Acta2-Cre-ERT2;Rosa26LacZ mice injected with Tamoxifen at 4 weeks and analysed 20 weeks later, on day 25 of the involution following second pregnancy (second Inv25). Scale bar = 0.87 mm. Arrows point to alveoli. BV, blood vessel; D, mammary duct.

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Prater, M., Petit, V., Alasdair Russell, I. et al. Mammary stem cells have myoepithelial cell properties. Nat Cell Biol 16, 942–950 (2014) doi:10.1038/ncb3025

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