Advertisement

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

  • Yurika Sakai
  • Ruka Miyake
  • Tatsuya Shimizu
  • Tadaaki Nakajima
  • Teruyo Sakakura
  • Yasuhiro TomookaEmail author
Article

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.

Keywords

Stem cell Clonal cell line Mammary gland p53 deficiency 

Notes

Acknowledgments

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

Funding information

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

Supplementary material

11626_2019_406_MOESM1_ESM.xlsx (10 kb)
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)
11626_2019_406_MOESM2_ESM.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)
11626_2019_406_MOESM3_ESM.xlsx (11 kb)
ESM 1 (XLSX 10 kb)
11626_2019_406_Fig9_ESM.png (24 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)

11626_2019_406_MOESM4_ESM.TIF
High Resolution Image (TIF 44 kb)
11626_2019_406_Fig10_ESM.png (703 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)

11626_2019_406_MOESM5_ESM.TIF
High Resolution Image (TIF 959 kb)
11626_2019_406_Fig11_ESM.png (629 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)

11626_2019_406_MOESM6_ESM.TIF
High Resolution Image (TIF 841 kb)
11626_2019_406_Fig12_ESM.png (191 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)

11626_2019_406_MOESM7_ESM.TIF
High Resolution Image (TIF 222 kb)
11626_2019_406_Fig13_ESM.png (958 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)

11626_2019_406_MOESM8_ESM.TIF
High Resolution Image (TIF 1282 kb)

References

  1. 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 3:429–441CrossRefGoogle Scholar
  2. 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 31:1857–1867CrossRefGoogle Scholar
  3. 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 61:53–60CrossRefGoogle Scholar
  4. 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 19:515–520PubMedGoogle Scholar
  5. 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 38:2103–2111PubMedGoogle Scholar
  6. 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 293:414–425CrossRefGoogle Scholar
  7. 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 356:215–221CrossRefGoogle Scholar
  8. 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 6:605–615CrossRefGoogle Scholar
  9. 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 1:555–567CrossRefGoogle Scholar
  10. 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 16:693–706CrossRefGoogle Scholar
  11. 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 33:668–671CrossRefGoogle Scholar
  12. 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 46:214–225CrossRefGoogle Scholar
  13. Horiuchi H, Itoh M, Pleasure DE, Tomooka Y (2005) Characterization of a multipotent neural progenitorcell line FBD-103a and subclones. Brain Res 1066:24–36CrossRefGoogle Scholar
  14. 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 79:4074–4077CrossRefGoogle Scholar
  15. Inman JL, Robertson C, Mott JD, Bissell MJ (2015) Mammary gland development: cell fate specification, stem cells and the microenvironment. Development 142:1028–1042CrossRefGoogle Scholar
  16. 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 53:3374–3381PubMedGoogle Scholar
  17. 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 35:425–444CrossRefGoogle Scholar
  18. 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 142:893–904CrossRefGoogle Scholar
  19. 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 66:6063–6071CrossRefGoogle Scholar
  20. 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 105:1680–1685CrossRefGoogle Scholar
  21. 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:e1001630CrossRefGoogle Scholar
  22. 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 18:1094–1099CrossRefGoogle Scholar
  23. Roarty K, Rosen JM (2010) Wnt and mammary stem cells: hormones can't fly wingless. Curr Opin Pharmacol 10:643–649CrossRefGoogle Scholar
  24. Sakakura T (1987) Mammary embryogenesis. In: Neville MC, Daniel CW (eds) The mammary gland; development, regulation and function. Plenum Press, New York, pp 37–66CrossRefGoogle Scholar
  25. Sakakura T, Nishizuka Y, Dawe CJ (1976) Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland. Science 194:1439–1441CrossRefGoogle Scholar
  26. 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 63:733–736CrossRefGoogle Scholar
  27. 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 542:313–319CrossRefGoogle Scholar
  28. 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 439:84–88CrossRefGoogle Scholar
  29. 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 10:183–197CrossRefGoogle Scholar
  30. 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 439:993–997CrossRefGoogle Scholar
  31. 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 46:457–468CrossRefGoogle Scholar
  32. 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 38:547–556CrossRefGoogle Scholar
  33. 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 29:119–127CrossRefGoogle Scholar
  34. Tomooka Y, Bern HA (1982) Growth of mouse mammary glands after neonatal sex hormone treatment. J Natl Cancer Inst 69:1347–1352PubMedGoogle Scholar
  35. 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 8:3313–3322PubMedGoogle Scholar
  36. 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 39:146–156Google Scholar
  37. 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 107:4601–4606CrossRefGoogle Scholar
  38. 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 11:387–400CrossRefGoogle Scholar
  39. 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 517:81–84CrossRefGoogle Scholar
  40. 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 6:568–577CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Yurika Sakai
    • 1
  • Ruka Miyake
    • 1
  • Tatsuya Shimizu
    • 1
  • Tadaaki Nakajima
    • 1
    • 2
  • Teruyo Sakakura
    • 3
  • Yasuhiro Tomooka
    • 1
    Email author
  1. 1.Department of Biological Science and TechnologyTokyo University of ScienceTokyoJapan
  2. 2.Institute of Industrial ScienceThe University of TokyoTokyoJapan
  3. 3.Research Center for Matrix BiologyMie UniversityTsu CityJapan

Personalised recommendations