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


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.


Stem cell Clonal cell line Mammary gland p53 deficiency 



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)

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)

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)

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)

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)

High Resolution Image (TIF 1282 kb)


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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

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