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A clonal stem cell line established from a mouse mammary placode with ability to generate functional mammary glands

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Abstract

The mammary gland develops from the placode at ectodermal invagination. The rudimentary parenchyma (mammary bud) develops mammary trees and alveolar structures, suggesting that the mammary bud consists of stem/progenitor cells. Here, we established a clonal stem cell line from a mammary bud of a p53 null female embryo at day 14.5. FP5-3-1 line was a homogeneous cell population with polygonal epithelial morphology and spontaneously became heterogeneous during passages. Recloning gave rise to four sublines; three sublines have basal epithelial property and one subline has luminal epithelial property. The former sublines generate functional mammary glands when injected into cleared fat pads and the latter subline does not. The cell lines also express many stemness-related genes. The clonal cell lines established in the present study are shown to be mammary stem cells and not tumorigenic. They provide useful models for normal and tumor biology of the mammary gland in vivo and in vitro.

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References

  • Bouras T, Pal B, Vaillant F, Harburg G, Asselin-Labat M-L, Oakes SR, Lindeman GJ, Visvader JE (2008) Notch signaling regulates mammary stem cell function and luminal cell-fate commitment. Cell Stem Cell 3:429–441

    Article  CAS  Google Scholar 

  • Chiche A, Moumen M, Petit V, Jonkers J, Medina D, Deugnier MA, Faraldo MM, Glukhova MA (2013) Somatic loss of p53 leads to stem/progenitor cell amplification in both mammary epithelial compartments, basal and luminal. Stem Cells 31:1857–1867

    Article  CAS  Google Scholar 

  • Daniel CW, DeOme KB, Young JT, Blair PB, Faulkin L Jr (1968) The in vivo life span of normal and preneoplastic mouse mammary glands: a serial transplantation study. Natl Acad Sci U S A 61:53–60

    Article  CAS  Google Scholar 

  • DeOme KB, Faulkin LJ, Bern HA, Blair PB (1959) Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19:515–520

    CAS  PubMed  Google Scholar 

  • DeOme KB, Miyamoto MJ, Osborn RC, Guzman RC, Lum K (1978) Detection of inapparent nodule-transformed cells in the mammary gland tissues of virgin female BALB/cfC3H mice. Cancer Res 38:2103–2111

    CAS  PubMed  Google Scholar 

  • Deugnier MA, Faraldo MM, Teulière J, Thiery JP, Medin D, Glukhova MA (2006) Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the comma-Dβ cell line. Dev Biol 293:414–425

    Article  CAS  Google Scholar 

  • Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, Butel JS et al (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356:215–221

    Article  CAS  Google Scholar 

  • Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS (2004) Role of notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 6:605–615

    Article  Google Scholar 

  • Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567

    Article  CAS  Google Scholar 

  • Gudjonsson T, Villadsen R, Nielsen HL, Ronnov-Jessen L, Bissell MJ, Petersen OW (2002) Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. Genes Dev 16:693–706

    Article  CAS  Google Scholar 

  • Hanazono M, Hirabayashi Z, Tomisawa H, Aizawa S, Tomooka Y (1997) Establishment of uterine cell lines from p53-deficient mice. In Vitro Cell Dev Biol Anim 33:668–671

    Article  CAS  Google Scholar 

  • Hanazono M, Nozawa R, Itakura R, Aizawa S, Tomooka Y (2001) Establishment of an androgen-responsive prostatic cell line “PEA5” from a p53-deficient mouse. Prostate 46:214–225

    Article  CAS  Google Scholar 

  • Horiuchi H, Itoh M, Pleasure DE, Tomooka Y (2005) Characterization of a multipotent neural progenitorcell line FBD-103a and subclones. Brain Res 1066:24–36

    Article  CAS  Google Scholar 

  • Imagawa W, Tomooka Y, Nandi S (1982) Serum-free growth of normal and tumor mouse mammary epithelial cells in primary culture. Natl Acad Sci U S A 79:4074–4077

    Article  CAS  Google Scholar 

  • Inman JL, Robertson C, Mott JD, Bissell MJ (2015) Mammary gland development: cell fate specification, stem cells and the microenvironment. Development 142:1028–1042

    Article  CAS  Google Scholar 

  • Jerry DJ, Ozbun MA, Kittrell FS, Lane DP, Medina D, Bute JS (1993) Mutations in p53 are frequent in the preneoplastic stage of mouse mammary tumor development. Cancer Res 53:3374–3381

    CAS  PubMed  Google Scholar 

  • Lin MC, Chen SY, Tsai HM, He PL, Lin YC, Herschman H et al (2017) PGE2/EP4 signaling controls the transfer of the mammary stem cell state by lipid rafts in extracellular vesicles. Stem Cells 35:425–444

    Article  CAS  Google Scholar 

  • Lindley LE, Curtis KM, Sanchez-Mejias A, Rieger ME, Robbins DJ, Briege KJ (2015) The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage. Development 142:893–904

    Article  CAS  Google Scholar 

  • Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, Suri P, Wicha MS (2006) Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 66:6063–6071

    Article  CAS  Google Scholar 

  • Liu S, Ginestier C, Charafe-Jauffret E, Foco H, Kleer CG, Merajver SD, Dontu G, Wicha MS (2008) BRCA1 regulates human mammary stem/progenitor cell fate. Natl Acad Sci U S A 105:1680–1685

    Article  CAS  Google Scholar 

  • Makarem M, Kannan N, Nguyen LV, Knapp DJ, Balani S, Prater MD et al (2013) Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells. PLoS Biol 11:e1001630

    Article  CAS  Google Scholar 

  • Pietersen AM, Evers B, Prasad AA, Tanger E, Cornelissen-Steijger P, Jonkers J, van Lohuizen M (2008) Bmi1 regulates stem cells and proliferation and differentiation of committed cells in mammary epithelium. Curr Biol 18:1094–1099

    Article  CAS  Google Scholar 

  • Roarty K, Rosen JM (2010) Wnt and mammary stem cells: hormones can't fly wingless. Curr Opin Pharmacol 10:643–649

    Article  CAS  Google Scholar 

  • Sakakura T (1987) Mammary embryogenesis. In: Neville MC, Daniel CW (eds) The mammary gland; development, regulation and function. Plenum Press, New York, pp 37–66

    Chapter  Google Scholar 

  • Sakakura T, Nishizuka Y, Dawe CJ (1976) Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland. Science 194:1439–1441

    Article  CAS  Google Scholar 

  • Sakakura T, Nishizuka Y, Dawe CJ (1979) Capacity of mammary fat pads of adult C3H/HeMs mice to interact morphogenetically with fetal mammary epithelium. J Natl Cancer Inst 63:733–736

    Article  CAS  Google Scholar 

  • Scheele CL, Hannezo E, Muraro MJ, Zomer A, Langedijk NSM, van Oudenaarden A et al (2017) Identity and dynamics of mammary stem cells during branching morphogenesis. Nature 542:313–319

    Article  CAS  Google Scholar 

  • Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE (2006) Generation of a functional mammary gland from a single stem cell. Nature 439:84–88

    Article  CAS  Google Scholar 

  • Spike BT, Engle DD, Lin JC, Cheung SK, La J, Wahl GM (2012) A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. Cell Stem Cell 10:183–197

    Article  CAS  Google Scholar 

  • Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D et al (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439:993–997

    Article  CAS  Google Scholar 

  • Takahashi C, Yoshida H, Komine A, Nakao K, Tsuji T, Tomooka Y (2010) Newly established cell lines from mouse oral epithelium regenerate teeth when combined with dental mesenchyme. In Vitro Cell Dev Biol Anim 46:457–468

    Article  CAS  Google Scholar 

  • Tanahashi K, Shibahara S, Ogawa M, Hanazono M, Aizawa S, Tomooka Y (2002) Establishment and characterization of clonal cell lines from the vagina of p53-deficient young mice. In Vitro Cell Biol Anim 38:547–556

    Article  Google Scholar 

  • Tao L, Roberts AL, Karen A, Dunphy KA, Carol BC, Haoheng YH et al (2011) Repression of mammary stem/progenitor cells by p53 is mediated by notch and separable from apoptotic activity. Stem Cells 29:119–127

    Article  CAS  Google Scholar 

  • Tomooka Y, Bern HA (1982) Growth of mouse mammary glands after neonatal sex hormone treatment. J Natl Cancer Inst 69:1347–1352

    CAS  PubMed  Google Scholar 

  • Tsukada T, Tomooka Y, Takai S, Ueda Y, Nishikawa S, Yagi T et al (1993) Enhanced proliferative potential in culture of cells from p53-deficient mice. Oncogene 8:3313–3322

    CAS  PubMed  Google Scholar 

  • Umezu T, Hanazono M, Aizawa S, Tomooka Y (2003) Characterization of newly established clonal oviductal cell lines and differentiation of gene expression. In Vitro Cell Biol Anim 39:146–156

    CAS  Google Scholar 

  • Umezu T, Yamanouchi H, Iida Y, Miura M, Tomooka Y (2010) Follistatin-like-1, a diffusible mesenchymal factor determines the fate of epithelium. Natl Acad Sci U S A 107:4601–4606

    Article  CAS  Google Scholar 

  • Van Amerongen R, Bowman AN, Nussel R (2012) Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11:387–400

    Article  Google Scholar 

  • Wang D, Cai C, Dong X, Yu QC, Zhang XO, Li YL et al (2015) Identification of multipotent mammary stem cells by protein C receptor expression. Nature 517:81–84

    Article  CAS  Google Scholar 

  • Zeng YA, Nusse R (2010) Wnt proteins are self-renewing factors for mammary stem cells and promote their long-term expansion in culture. Cell Stem Cell 6:568–577

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The funding body had no role in the design or execution of the study.

Funding

This work was supported by a Grants for Support of the Promotion of Research at Tokyo University of Science (to YT and TN).

Author information

Author notes

  1. Yurika Sakai, Ruka Miyake, Tatsuya Shimizu and Tadaaki Nakajima contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan

    Yurika Sakai, Ruka Miyake, Tatsuya Shimizu, Tadaaki Nakajima & Yasuhiro Tomooka

  2. Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan

    Tadaaki Nakajima

  3. Research Center for Matrix Biology, Mie University, 2-174 Edobashi, Tsu City, Mie, 514-8507, Japan

    Teruyo Sakakura

Authors
  1. Yurika Sakai
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  2. Ruka Miyake
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  3. Tatsuya Shimizu
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  4. Tadaaki Nakajima
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  5. Teruyo Sakakura
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  6. Yasuhiro Tomooka
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Corresponding author

Correspondence to Yasuhiro Tomooka.

Additional information

Editor: Tetsuji Okamoto

Electronic supplementary material

Table S1

Primer used for RT-PCR. Primers used for RT-PCR for stemness-related genes and for genomic PCR for analysis of sex origin were listed. (XLSX 10 kb)

Table S2

Outgrowth of mammary cells. Cells (1.0 × 106) were injected into cleared fat pads of CD-1 mice or nude mice. Successful outgrowth rate (%) and numbers of transplantation in parentheses are presented. *: injection without Matrigel. **: Cells were mixed at even ratio. (XLSX 9 kb)

ESM 1

(XLSX 10 kb)

Figure S1

Evidence of female origin of FP5–1-3 line. FP5–1-3 cells were subjected to RT-PCR analysis of gSry as a marker of male. (PNG 23 kb)

High Resolution Image (TIF 44 kb)

Figure S2

Outgrowth of sublines in combination transplanted into cleared fat pads. Whole mount iron hematoxylin staining of mammary glands derived from cells of O1 + L3 lines (a-1), O1 + M5 lines (b-1), L3 + M5 lines (c-1) and O1 + L2 + M5 lines (d-1) in cleared fat pads. Immunohistochemistry of sections of mammary ducts for KRT8 (a-2, b-2, c-2, d-2) and SMA (a-3, b-3, c-3, d-3). Scale bar; 1.5 mm (a-1, b-1, c-1, d-1), 50 μm (a-2, 3, b-2, 3, c-2, 3, d-2, 3). (PNG 703 kb)

High Resolution Image (TIF 959 kb)

Figure S3

Differentiation of outgrowth of O1 and M5 cells in combination transplanted into cleared fat pads. Cells of O1 and M5 lines in combination were transplanted into cleared fat pads and the recipients were mated and sacrificed after labor. Generated mammary gland were stained with iron hematoxylin (a-1, a-2; high magnification). Immunohistochemistry of sections of ductal and alveolar structures for KRT8 (marker for luminal epithelial cells)/LTF (milk protein) (b-1, c-1) and SMA (marker for myoepithelial cells)/LTF (b-2, c-2). Scale bar; 3 mm (a-1), 1.5 mm (a-2), 50 μm (b, c). (PNG 628 kb)

High Resolution Image (TIF 841 kb)

Figure S4

Effects of media conditioned by sublines on expression of stemness-related genes. RT-PCR analysis for Procr, Aldh1a3 and Axin2 in O1 cells cultured in medium conditioned by O1 cell (control), O1 cells cultured in medium conditioned by M5 cell, M5 cells cultured in medium conditioned by M5 cell (control), M5 cells cultured in medium conditioned by O1 cell and primary mammary epithelial cells (a). β-actin was chosen as internal standard. The results were semi-quantitatively presented (b, value of primary epithelial cells = 1). *: p < 0.05. (PNG 191 kb)

High Resolution Image (TIF 222 kb)

Figure S5

3D growth of sublines in vitro. O1 (a-1), L3 (a-2) and M5 cells (a-3) were cultured in Matrigel with mitomycin C treated mesenchymal cells (a cell line co-established from a mammary placode). Sections of 3D cultures of O1 (b-1), L3 (b-2) and M5 cells (b-3) were stained with HE. Scale bar; 200 μm (a), 50 μm (b). (PNG 958 kb)

High Resolution Image (TIF 1282 kb)

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Sakai, Y., Miyake, R., Shimizu, T. et al. A clonal stem cell line established from a mouse mammary placode with ability to generate functional mammary glands. In Vitro Cell.Dev.Biol.-Animal 55, 861–871 (2019). https://doi.org/10.1007/s11626-019-00406-8

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