Quantifying Epithelial Early Common Progenitors from Long‐Term Primary or Cell Line Sphere Culture

Flora Clément1, Helen He Zhu2, Wei‐Qiang Gao2, Emmanuel Delay3, Véronique Maguer‐Satta4

1 Université de Lyon, Université Lyon 1, Lyon, 2 School of Biomedical Engineering & Med‐X Research Institute, Shanghai Jiao Tong University, Shanghai, 3 Centre Léon Bérard, Lyon, 4 Department of Tumor Escape Signaling, Centre de Recherche en Cancérologie de Lyon, Lyon
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.7
DOI:  10.1002/9780470151808.sc01e07s35
Online Posting Date:  November, 2015
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Abstract

Here, a protocol to quantify epithelial early common progenitor/stem cells grown as spheres in non‐adherent culture conditions is described. This protocol is based on the combination of two functional tests: the sphere assay to maintain and enrich early progenitor/stem cells, and the epithelial colony‐forming cells (E‐CFC) assay to identify and quantify further differentiated epithelial progenitors. Primary spheres mainly contain progenitors and rare stem/early common progenitor cells while secondary and tertiary spheres contain progenitor cells derived from the early common progenitor/stem cell population maintained through passages and partially differentiated. Spheres are enzymatically and mechanically dissociated; the derived cells are subsequently plated on irradiated NIH‐3T3 fibroblasts for further processing, as in the E‐CFC assay. The principle of this assay is to quantify the number of epithelial colonies generated by cells present in the different sequential spheres. This assay has therefore been named the early common progenitor‐derived colonies assay (ECP‐DC). © 2015 by John Wiley & Sons, Inc.

Keywords: human stem cells; in vitro quantification; spheres; epithelial colonies; cell lines

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintenance of Epithelial Early Common Progenitors and Stem Cells by Sphere Culture
  • Basic Protocol 2: Early Common Progenitor‐Derived Colony (ECP‐DC) Assay
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Maintenance of Epithelial Early Common Progenitors and Stem Cells by Sphere Culture

  Materials
  • Secondary or tertiary spheres derived from normal epithelial cell suspension obtained from digestion of human mammary tissue (Bachelard‐Cascales et al., ; Chapellier et al., ) or breast and prostate normal and tumoral cell lines
  • MEBM medium (Invitrogen) supplemented with 1 × B27 (Invitrogen), 20 ng/ml EGF (R&D Systems), 20 ng/ml bFGF (StemCell Technologies), 4 μg/ml heparin, and 100 U/ml penicillin/100 μg/ml streptomycin (Invitrogen)
  • 0.05% trypsin/0.025% EDTA (Invitrogen)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • Fetal bovine serum (FBS)
  • 100 U/ml penicillin/100 μg/ml streptomycin (Invitrogen)
  • Ultra‐low attachment plates (Corning Life Sciences)
  • Centrifuge
  • 37°C incubator
  • 15‐ml centrifuge tubes
  • 40‐μm nylon membrane (BD Biosciences/Pharmingen)
  • Hemacytometer

Basic Protocol 2: Early Common Progenitor‐Derived Colony (ECP‐DC) Assay

  Materials
  • Mitotically inactivated (30 Gray irradiation) NIH‐3T3 mouse fibroblasts (ATCC #CRL‐1658)
  • DMEM supplemented with 10% FBS
  • Secondary/tertiary sphere‐derived single‐cell suspension (see protocol 1)
  • Epicult B medium (StemCell Technologies) supplemented with 5% FBS or 0.1% BSA and 100 U/ml penicillin/100 μg/ml streptomycin (Invitrogen)
  • Cell line–specific medium
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • Methanol, ice cold
  • Wright dye (RAL; http://www.reactifs‐ral.fr)
  • 6‐ to 24‐well tissue culture plates (Falcon, Becton Dickinson)
  • 37°C incubator
  • Aspirator
  • Microscope
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Figures

Videos

Literature Cited

Literature Cited
  Bachelard‐Cascales, E., Chapellier, M., Delay, E., Pochon, G., Voeltzel, T., Puisieux, A., Caron, d. F., and Maguer‐Satta, V. 2010. The CD10 enzyme is a key player to identify and regulate human mammary stem cells. Stem Cells 28:1081‐1088. doi: 10.1002/stem.435.
  Chapellier, M., Bachelard‐Cascales, E., Schmidt, X., Clement, F., Treilleux, I., Delay, E., Jammot, A., Menetrier‐Caux, C., Pochon, G., Besancon, R., Voeltzel, T., Caron de Fromentel, C., Caux, C., Blay, J.Y., Iggo, R., and Maguer‐Satta, V. 2015. Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying bmpr1b cell response. Stem Cell Rep. 4:239‐254. doi: 10.1016/j.stemcr.2014.12.007.
  Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J., and Wicha, M.S. 2003. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes. Dev. 17:1253‐1270. doi: 10.1101/gad.1061803.
  Garraway, I.P., Sun, W., Tran, C.P., Perner, S., Zhang, B., Goldstein, A.S., Hahm, S.A., Haider, M., Head, C.S., Reiter, R.E., Rubin, M.A., and Witte, O.N. 2010. Human prostate sphere‐forming cells represent a subset of basal epithelial cells capable of glandular regeneration in vivo. Prostate 70:491‐501.
  Maguer‐Satta, V., Chapellier, M., Delay, E., and Bachelard‐Cascales, E. 2011. CD10: A tool to crack the role of stem cells in breast cancer. Proc. Natl. Acad. Sci. U.S.A. 108:E1264. doi: 10.1073/pnas.1116567108.
  Pfeiffer, M.J. and Schalken, J.A. 2010. Stem cell characteristics in prostate cancer cell lines. Eur. Urol. 57:246‐254. doi: 10.1016/j.eururo.2009.01.015.
  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. doi: 10.1111/j.1432‐0436.1998.00201.x.
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