Preparation of Epithelial and Mesenchymal Stem Cells from Murine Mammary Gland

Ian Guest1, Zoran Ilic1, Jun Ma1

1 Department of Translational Medicine, Wadsworth Laboratories, New York State Department of Health, Albany, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 22.3
DOI:  10.1002/0471140856.tx2203s50
Online Posting Date:  November, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44, and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumor development. Curr. Protoc. Toxicol. 50:22.3.1‐22.3.15. © 2011 by John Wiley & Sons, Inc.

Keywords: mammary gland (tumor); cancer stem cell; epithelial; mesenchymal; polyoma middle T antigen transgenic mouse

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Flow Cytometric Sorting of Epithelial and Mesenchymal Stem Cells from Mammary Gland Tumors of PyVT Mice
  • Alternate Protocol 1: Alternate Method for Mesenchymal Cell Isolation from Bone Marrow
  • Support Protocol 1: Culture of Epithelial and Mesenchymal Cells
  • Support Protocol 2: Cryopreservation of Cells
  • Support Protocol 3: Clearance of Mammary Gland Fat Pad and Transplantation of Cells
  • Support Protocol 4: Ectopic Transplantation of Isolated Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation and Flow Cytometric Sorting of Epithelial and Mesenchymal Stem Cells from Mammary Gland Tumors of PyVT Mice

  Materials
  • Mice: FVB/N‐Tg(MMTV‐PyVT)634Mul/J (PyVT) (Jackson Laboratories, stock no. 002374)
  • CO 2 source
  • 70% (v/v) ethanol (see recipe)
  • Betadine solution
  • Dulbecco's modified Eagle's medium (DMEM; see recipe)
  • 0.25% (w/v) trypsin (see recipe), sterile
  • Fetal bovine serum (FBS; see recipe)
  • Trypan blue solution (see recipe)
  • Staining medium (see recipe): Hank's balanced salt solution with and without 2% heat‐inactivated FBS, cold
  • Antibodies (Biolegend): anti‐CD24‐fluorescein isothiocyanate (cat. no. 101805), anti‐CD44‐Pacific Blue (cat. no. 103019), anti‐CD49f‐AlexaFluor 647 (cat. no. 313609), anti‐CD45‐phycoerythrin (cat. no. 103105), anti‐CD31‐phycoerythrin (cat. no. 102407), anti‐Ter119‐allophycocyanin (cat. no. 116211), anti‐Sca1‐phycoerythrin (cat. no. 108109)
  • Surgical scissors, tweezers, and scalpels
  • Disposable 35‐ and 100‐mm petri dishes
  • Sterile 1‐, 5‐, 10‐, and 25‐ml sterile serological pipets
  • 37°C, CO 2‐regulated tissue culture incubator
  • Sterile 70‐µm cell strainers
  • 15‐ and 50‐ml screw‐capped sterile centrifuge tubes
  • Refrigerated centrifuge
  • Hemacytometer
  • Flow cytometer with sorting capacity (e.g., FACSVantage, Becton Dickinson)

Alternate Protocol 1: Alternate Method for Mesenchymal Cell Isolation from Bone Marrow

  • FVB mice (Jackson Laboratories, stock no. 001800)
  • Red blood cell lysing buffer (Sigma; store 2 years at room temperature)
  • Mesencult complete medium (see recipe)
  • Anti‐CD11b‐FITC (Biolegend, cat. no. 101205)
  • Bone scissors
  • 3‐ml syringes with 25‐G needles
  • Tissue culture flasks (25‐, 75‐, and 175‐ml filter‐top flasks)

Support Protocol 1: Culture of Epithelial and Mesenchymal Cells

  Materials
  • Isolated and sorted cells (see protocol 1 or protocol 2)
  • Dulbecco's Modified Eagle's Medium (DMEM; see recipe) or Mesencult complete medium (see recipe)
  • 10% FBS (see recipe)
  • Epidermal growth factor (see recipe)
  • Insulin (see recipe)
  • HBSS (see recipe)
  • 37°C, 5% CO 2 tissue culture incubator
  • Sterile pipets (1‐, 5‐, 10‐, and 25‐ml serological pipettes)
  • Sterile 0.22‐µm membrane filters (Millipore)

Support Protocol 2: Cryopreservation of Cells

  Materials
  • Epithelial or mesenchymal cells (70% confluent)
  • DMEM medium (see recipe) or Mesencult complete medium (see recipe)
  • Cryopreservation medium (see recipe)
  • Refrigerated centrifuge
  • 1‐ml cryotubes
  • Freezing container (e.g., Mr Frosty, Nalgene)

Support Protocol 3: Clearance of Mammary Gland Fat Pad and Transplantation of Cells

  Materials
  • FVB mice (must be no older than 3 and 1/2 weeks)
  • Ketamine/xylazine mix (see recipe)
  • 70% ethanol (see recipe)
  • Betadine
  • Prepared cells ready for transplant in PBS (see recipe)
  • Surgical instruments
  • Cauterizer (e.g., Roboz Surgical Instrument, model RS‐320)
  • 25‐ to 50‐µl microsyringes (Hamilton) and 27‐G needles
  • Wound closing clips and applicator (Clay Adams Autoclips)

Support Protocol 4: Ectopic Transplantation of Isolated Cells

  Materials
  • Mice
  • 70% ethanol
  • Cells
  • 1‐ml syringe and 26‐G, 5/8‐in. needle
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Al‐Hajj, M., Wicha, M.S., Benito‐Hernandez, A., Morrison, S.J., and Clarke, M.F. 2003. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 100:3983‐3988.
   Anjos‐Afonso, F. and Bonnet, D. 2008. Isolation, culture and differentiation potential of mouse marrow stromal cells. Curr. Protoc. Stem Cell Biol. 7:2B.3.1‐2B.3.11.
   Baumann, P., Cremers, N., Kroese, F., Orend, G., Chiquet‐Ehrismann, R., Uede, T., Yagita, H., and Sleeman, J.P. 2005. CD24 expression causes the acquisition of multiple cellular properties associated with tumor growth and metastasis. Cancer Res. 65:10783‐10793.
   Bircan, S., Kapucuoglu, N., Baspinar, S., Inan, G., and Candir, O. 2006. CD24 expression in ductal carcinoma in situ and invasive ductal carcinoma of breast: An immunohistochemistry‐based pilot study. Pathol. Res. Pract. 202:569‐576.
   Bohl, S.R., Pircher, A., and Hilbe, W. 2011. Cancer stem cells: Characteristics and their potential role for new therapeutic strategies. Onkologie 34:269‐274.
   Cardiff, R.D. and Wellings, S.R. 1999. The comparative pathology of human and mouse mammary glands. J. Mammary Gland Biol. Neoplasia 4:105‐122.
   Cho, R.W., Wang, X., Diehn, M., Shedden, K., Chen, G.Y., Sherlock, G., Gurney, A., Lewicki, J., and Clarke, M.F. 2008. Isolation and molecular characterization of cancer stem cells in MMTV‐Wnt‐1 murine breast tumors. Stem Cells 26:364‐371.
   Clarke, R.B. 2005. Isolation and characterization of human mammary stem cells. Cell Prolif. 38:375‐386.
   Collins, M.D. and Gibson, G.R. 1999. Probiotics, prebiotics, and synbiotics: Approaches for modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 69:1052S‐1057S.
   Dalerba, P., Dylla, S.J., Park, I.K., Liu, R., Wang, X., Cho, R.W., Hoey, T., Gurney, A., Huang, E.H., Simeone, D.M., Shelton, A.A., Parmiani, G., Castelli, C., and Clarke, M.F. 2007. Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl. Acad. Sci. U.S.A. 104:10158‐10163.
   Daniel, C.W. and Silberstein, G.B. 1987. Postnatal development of the rodent mammary gland. In The Mammary Gland. Development, Regulation and Function. (M.C. Neville, and C.W. Daniel, eds.) pp. 1‐35. Plenum, New York.
   Daniel, C.W., De Ome, K.B., Young, J.T., Blair, P.B., and Faulkin, L.J. Jr. 1968. The in vivo life span of normal and preneoplastic mouse mammary glands: A serial transplantation study. Proc. Natl. Acad. Sci. U.S.A. 61:53‐60.
   Deome, K.B., Faulkin, L.J. Jr., Bern, H.A., and Blair, P.B. 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.
   Fillmore, C.M. and Kuperwasser, C. 2008. Human breast cancer cell lines contain stem‐like cells that self‐renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res. 10:R25.
   Fogel, M., Friederichs, J., Zeller, Y., Husar, M., Smirnov, A., Roitman, L., Altevogt, P., and Sthoeger, Z.M. 1999. CD24 is a marker for human breast carcinoma. Cancer Lett. 143:87‐94.
   Franco, O.E., Shaw, A.K., Strand, D.W., and Hayward, S.W. 2010. Cancer associated fibroblasts in cancer pathogenesis. Semin. Cell Dev. Biol. 21:33‐39.
   Friedenstein, A.J., Petrakova, K.V., Kurolesova, A.I., and Frolova, G.P. 1968. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6:230‐247.
   Goldstein, R.H., Reagan, M.R., Anderson, K., Kaplan, D.L., and Rosenblatt, M. 2010. Human bone marrow‐derived MSCs can home to orthotopic breast cancer tumors and promote bone metastasis. Cancer Res. 70:10044‐10050.
   Guest, I., Ilic, Z., Ma, J., Grant, D., Glinsky, G., and Sell, S. 2010. Direct and indirect contribution of bone marrow‐derived cells to cancer. Int. J. Cancer 126:2308‐2318.
   Halpern, J.L., Kilbarger, A., and Lynch, C.C. 2011. Mesenchymal stem cells promote mammary cancer cell migration in vitro via the CXCR2 receptor. Cancer Lett. 308:91‐99.
   Hermann, P.C., Bhaskar, S., Cioffi, M., and Heeschen, C. 2010. Cancer stem cells in solid tumors. Semin Cancer Biol. 20:77‐84.
   Hwang, R.F., Moore, T., Arumugam, T., Ramachandran, V., Amos, K.D., Rivera, A., Ji, B., Evans, D.B., and Logsdon, C.D. 2008. Cancer‐associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res. 68:918‐926.
   Jacobsen, B.M., Harrell, J.C., Jedlicka, P., Borges, V.F., Varella‐Garcia, M., and Horwitz, K.B. 2006. Spontaneous fusion with, and transformation of mouse stroma by, malignant human breast cancer epithelium. Cancer Res. 66:8274‐8279.
   Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz‐Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W.C., Largaespada, D.A., and Verfaillie, C.M. 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41‐49.
   Johnsen, H.E., Kjeldsen, M.K., Urup, T., Fogd, K., Pilgaard, L., Boegsted, M., Nyegaard, M., Christiansen, I., Bukh, A., and Dybkaer, K. 2009. Cancer stem cells and the cellular hierarchy in haematological malignancies. Eur. J. Cancer 45:194‐201.
   LaBarge, M.A., Petersen, O.W., and Bissell, M.J. 2007. Of microenvironments and mammary stem cells. Stem Cell Rev. 3:137‐146.
   Li, C., Heidt, D.G., Dalerba, P., Burant, C.F., Zhang, L., Adsay, V., Wicha, M., Clarke, M.F., and Simeone, D.M. 2007. Identification of pancreatic cancer stem cells. Cancer Res. 67:1030‐1037.
   Lim, E., Wu, D., Pal, B., Bouras, T., Asselin‐Labat, M.L., Vaillant, F., Yagita, H., Lindeman, G.J., Smyth, G.K., and Visvader, J.E. 2010. Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways. Breast Cancer Res. 12:R21.
   Liu, S., Ginestier, C., Ou, S.J., Clouthier, S.G., Patel, S.H., Monville, F., Korkaya, H., Heath, A., Dutcher, J., Kleer, C.G., Jung, Y., Dontu, G., Taichman, R., and Wicha, M.S. 2011. Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks. Cancer Res. 71:614‐624.
   Meyer, M.J., Fleming, J.M., Ali, M.A., Pesesky, M.W., Ginsburg, E., and Vonderhaar, B.K. 2009. Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell lines. Breast Cancer Res. 11:R82.
   Nieoullon, V., Belvindrah, R., Rougon, G., and Chazal, G. 2007. Mouse CD24 is required for homeostatic cell renewal. Cell Tissue Res. 329:457‐467.
   Phinney, D.G., Kopen, G., Isaacson, R.L., and Prockop, D.J. 1999. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J. Cell Biochem. 72:570‐585.
   Placencio, V.R., Li, X., Sherrill, T.P., Fritz, G., and Bhowmick, N.A. 2010. Bone marrow derived mesenchymal stem cells incorporate into the prostate during regrowth. PLoS One. 5:e12920.
   Rhodes, L.V., Muir, S.E., Elliott, S., Guillot, L.M., Antoon, J.W., Penfornis, P., Tilghman, S.L., Salvo, V.A., Fonseca, J.P., Lacey, M.R., Beckman, B.S., McLachlan, J.A., Rowan, B.G., Pochampally, R., and Burow, M.E. 2010. Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence. Breast Cancer Res. Treat. 121:293‐300.
   Sell, S. 2010. On the stem cell origin of cancer. Am. J. Pathol. 176:2584‐2594.
   Shackleton, M. 2010. Normal stem cells and cancer stem cells: Similar and different. Semin. Cancer Biol. 20:85‐92.
   Shackleton, M., Vaillant, F., Simpson, K.J., Stingl, J., Smyth, G.K., Asselin‐Labat, M.L., Wu, L., Lindeman, G.J., and Visvader, J.E. 2006. Generation of a functional mammary gland from a single stem cell. Nature 439:84‐88.
   Sleeman, J.P. and Cremers, N. 2007. New concepts in breast cancer metastasis: Tumor initiating cells and the microenvironment. Clin. Exp. Metastasis 24:707‐715.
   Sleeman, K.E., Kendrick, H., Ashworth, A., Isacke, C.M., and Smalley, M.J. 2006. CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non‐epithelial cells. Breast Cancer Res. 8:R7.
   Stingl, J. 2009. Detection and analysis of mammary gland stem cells. J. Pathol. 217:229‐241.
   Stingl, J., Eaves, C.J., Kuusk, U., and Emerman, J.T. 1998. Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. Differentiation 63:201‐213.
   Stingl, J., Eaves, C.J., Zandieh, I., and Emerman, J.T. 2001. Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res. Treat. 67:93‐109.
   Vigetti, D., Viola, M., Karousou, E., Rizzi, M., Moretto, P., Genasetti, A., Clerici, M., Hascall, V.C., De Luca, G., and Passi, A. 2008. Hyaluronan‐CD44‐ERK1/2 regulate human aortic smooth muscle cell motility during aging. J. Biol. Chem. 283:4448‐4458.
   Villadsen, R., Fridriksdottir, A.J., Ronnov‐Jessen, L., Gudjonsson, T., Rank, F., LaBarge, M.A., Bissell, M.J., and Petersen, O.W. 2007. Evidence for a stem cell hierarchy in the adult human breast. J. Cell Biol. 177:87‐101.
   Woodward, T.L., Xie, J.W., and Haslam, S.Z. 1998. The role of mammary stroma in modulating the proliferative response to ovarian hormones in the normal mammary gland. J. Mammary Gland Biol. Neoplasia 3:117‐131.
   Woodward, W.A., Chen, M.S., Behbod, F., and Rosen, J.M. 2005. On mammary stem cells. J. Cell Sci. 118:3585‐3594.
   Xouri, G. and Christian, S. 2010. Origin and function of tumor stroma fibroblasts. Semin. Cell Dev. Biol. 21:40‐46.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library