Isolation and Clonal Assay of Adult Lung Epithelial Stem/Progenitor Cells

Ivan Bertoncello1, Jonathan McQualter1

1 Lung Regeneration Laboratory, The Department of Pharmacology, University of Melbourne, Victoria, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2G.1
DOI:  10.1002/9780470151808.sc02g01s16
Online Posting Date:  January, 2011
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Abstract

Adult mouse lung epithelial stem/progenitor cells (EpiSPC) can be defined in vitro as epithelial colony‐forming units that are capable of self‐renewal, and which when co‐cultured with lung mesenchymal stromal cells (MSC) are able to give rise to differentiated progeny comprising mature lung epithelial cells. This unit describes a protocol for the prospective isolation and in vitro propagation and differentiation of adult mouse lung EpiSPC. The strategy used for selection of EpiSPC and MSC from adult mouse lung by enzymatic digestion and flow cytometry is based on the differential expression of CD45, CD31, Sca‐1, EpCAM, and CD24. The culture conditions required for the differentiation (co‐culture with MSC) and expansion (stromal‐free culture with FGF‐10 and HGF) of EpiSPC are described. Curr. Protoc. Stem Cell Biol. 16:2G.1.1‐2G.1.12. © 2011 by John Wiley & Sons, Inc.

Keywords: lung epithelium; stem cells; colony‐forming assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Epithelial Stem/Progenitor Cells and Mesenchymal Stromal Cells from Adult Mouse Lung
  • Basic Protocol 2: Organotypic Culture of Lung Epithelial Stem/Progenitor Cells (Differentiation Culture)
  • Basic Protocol 3: Expansion of Lung Epithelial Stem/Progenitor Cells in Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Epithelial Stem/Progenitor Cells and Mesenchymal Stromal Cells from Adult Mouse Lung

  Materials
  • Collagenase, type 1 (Worthington)
  • Phosphate buffered saline (PBS; Sigma, cat. no. P‐3813)
  • Adult mice (strain C57BL/6; 6 to 12 weeks; 18 to 22 g; male or female)
  • PBS with 2% (v/v) fetal bovine serum (FBS; JRH Biosciences, batch tested)
  • Nycoprep density gradient medium (1.077 g/cm3, 265 mOsm (see recipe)
  • Fluorochrome‐conjugated antibodies against:
    • EpCAM (clone G8.8)
    • CD24 (clone M1/69)
    • Sca‐1 (clone E13‐161.7)
    • CD45 (clone 30‐F11)
    • CD31 (clone 390)
  • Viability dye (4′,6‐diamidino‐2‐phenylindole (DAPI), propidium iodide (PI), or FluoroGold (Fluorochrome LLC, http://www.fluorochrome.com)
  • Dissecting equipment
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • 60‐mm‐diameter Petri dishes
  • Thermomixer (Eppendorf)
  • 18‐G and 21‐G needles
  • 5‐ml and 20‐ml syringes
  • 40‐µm cell strainer (BD Biosciences)
  • Refrigerated centrifuge (with capacity for 5‐ml, 15‐ml and 50‐ml tubes)
  • Sysmex KX‐21N cell counter (Sysmex Corporation; http://www.sysmex.com/) or hemacytometer counting chamber and trypan blue (unit 1.3)
  • Sterile mixing cannulas (Unomedical, cat. no. 500.11.012; http://www.unomedical.com)
  • 5‐ml FACS tubes with 35‐µm cell strainer caps (BD Biosciences), sterile
  • Flow cytometer
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ), determining viable cell concentration (unit 1.3), and flow cytometry (Robinson et al., )

Basic Protocol 2: Organotypic Culture of Lung Epithelial Stem/Progenitor Cells (Differentiation Culture)

  Materials
  • Epi‐CFU medium (see recipe)
  • Matrigel (standard concentration; BD Biosciences)
  • Adult lung EpiSPC (CD45negCD31negEpCAMhiCD24low) cells ( protocol 1)
  • Adult lung MSC (CD45negCD31negEpCAMnegSca‐1hi) cells ( protocol 1)
  • Refrigerated centrifuge
  • Millicell‐CM culture inserts (0.4‐µm membrane, 12‐mm diameter, hydrophilic PTFE, Millipore)
  • 24‐well flat‐bottom tissue culture plates
  • Triple‐mix incubator (5% v/v O 2, 10% v/v CO 2, 85% v/v N 2; see ), humidified
  • Additional reagents and equipment for determining viable cell concentration (unit 1.3)

Basic Protocol 3: Expansion of Lung Epithelial Stem/Progenitor Cells in Culture

  Materials
  • Epi‐CFU expansion medium (see recipe)
  • Matrigel (standard concentration; BD Biosciences)
  • Adult lung EpiSPC (CD45negCD31negEpCAMhiCD24low) cells ( protocol 1)
  • Phosphate buffered saline (PBS; Sigma, cat. no. P‐3813)
  • Enzymatic digestion cocktail (see recipe)
  • Refrigerated centrifuge
  • Millicell‐CM culture inserts (0.4‐µm membrane, 12‐mm diameter, hydrophilic PTFE, Millipore)
  • 24‐well flat‐bottom tissue culture plates
  • Triple‐mix incubator (5% v/v O 2, 10% v/v CO 2, 85% v/v N 2), humidified
  • 15‐ml conical tubes (e.g., BD Falcon) and 2‐ml microcentrifuge tubes
  • 21‐G needles
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Figures

Videos

Literature Cited

Literature Cited
   Alexander, C.M., Puchalski, J., Klos, K.S., Badders, N., Ailles, L., Kim, C.F., Dirks, P., and Smalley, M.J. 2009. Separating stem cells by flow cytometry: Reducing variability for solid tissues. Cell Stem Cell 5:579‐583.
   Alvarez, D.F., Helm, K., Degregori, J., Roederer, M., and Majka, S. 2010. Publishing flow cytometry data. Am. J. Physiol. Lung Cell Mol. Physiol. 298:L127‐L130.
   Bertoncello, I. and McQualter, J.L. 2010. Endogenous lung stem cells: What is their potential for use in regenerative medicine? Expert Rev. Respir. Med. 4:349‐362.
   Chen, H., Matsumoto, K., and Stripp, B.R. 2009. Bronchiolar progenitor cells. Proc. Am. Thorac. Soc. 6:602‐606.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Kim, C.F., Jackson, E.L., Woolfenden, A.E., Lawrence, S., Babar, I., Vogel, S., Crowley, D., Bronson, R.T., and Jacks, T. 2005. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823‐835.
   McQualter, J.L., Brouard, N., Williams, B., Baird, B.N., Sims‐Lucas, S., Yuen, K., Nilsson, S.K., Simmons, P.J., and Bertoncello, I. 2009. Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca‐1 positive cell fraction. Stem Cells 27:623‐633.
   McQualter, J.L., Yuen, K., Williams, B., and Bertoncello, I. 2010. Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung. Proc. Natl. Acad. Sci. U.S.A. 167:1414‐1419.
   Raiser, D.M. and Kim, C.F. 2009. Commentary: Sca‐1 and cells of the lung: A matter of different sorts. Stem Cells 27:606‐611.
   Reynolds, S.D., Shen, H., Reynolds, P.R., Betsuyaku, T., Pilewski, J.M., Gambelli, F., Di Giuseppe, M., Ortiz, L.A., and Stripp, B.R. 2007. Molecular and functional properties of lung SP cells. Am. J. Physiol. Lung Cell Mol. Physiol. 292:L972‐L983.
   Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P, Orfao, A., Rabinovitch, P., and Watkins, S. 2010. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
   Summer, R., Fitzsimmons, K., Dwyer, D., Murphy, J., and Fine, A. 2007. Isolation of an adult mouse lung mesenchymal progenitor cell population. Am. J. Respir. Cell Mol. Biol. 37:152‐159.
   Weiss, D.J., Kolls, J.K., Ortiz, L.A., Panoskaltsis‐Mortari, A., and Prockop, D.J. 2008. Stem cells and cell therapies in lung biology and lung diseases. Proc. Am. Thorac. Soc. 5:637‐667.
   Wion, D., Christen, T., Barbier, E.L., and Coles, J.A. 2009. PO(2) matters in stem cell culture. Cell Stem Cell 5:242‐243.
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