The Differentiation of Distal Lung Epithelium from Embryonic Stem Cells

Benjamin E. Van Vranken1, Helen J. Rippon2, Ali Samadikuchaksaraei3, Alan O. Trounson1, Anne E. Bishop2

1 Monash University, Clayton, 2 Imperial College London, London, England, 3 Iran University of Medical Sciences, Tehran
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.1
DOI:  10.1002/9780470151808.sc01g01s2
Online Posting Date:  July, 2007
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Abstract

The potential for embryonic stem (ES) cells to differentiate into cells with a distal lung epithelial phenotype has been demonstrated using different in vitro culture methods. Three separate protocols are described here that utilize both murine and human ES cells. The distal lung epithelial phenotype is induced through the use of embryonic distal lung mesenchyme in coculture systems with differentiating embryoid bodies or the use of soluble factors in defined media to maximize definitive endoderm formation and select and maintain the desired phenotype. Phenotypic analysis is demonstrated using immunocytochemistry and SP‐C promoter‐eGFP reporter gene expression in transgenic ES cells. These methods provide an increased efficiency of distal lung epithelial derivation from ES cells and, therefore, they provide the foundation for the development of a cell replacement product to treat chronic lung disease or a useful in vitro model for the study of lung disease and development. Curr. Protoc. Stem Cell Biol. 2:1G.1.1‐1G.1.22. © 2007 by John Wiley & Sons, Inc.

Keywords: embryonic stem cell; coculture; distal lung epithelium; differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Coculture of Differentiating Murine Embryoid Bodies with Murine Distal Embryonic Lung Mesenchyme
  • Support Protocol 1: Preparation of Neutralized Rat‐Tail Collagen Gel
  • Alternate Protocol 1: Indirect Coculture of Murine EBs and Murine Embryonic Distal Lung Mesenchyme
  • Basic Protocol 2: Derivation of Early Distal Lung Epithelial Progenitors from Murine ES Cells
  • Basic Protocol 3: Derivation of Distal Respiratory Epithelial Cells from Human ES Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Coculture of Differentiating Murine Embryoid Bodies with Murine Distal Embryonic Lung Mesenchyme

  Materials
  • Undifferentiated murine ES cell culture (feeder‐free;) in gelatinized (see recipe) 25‐cm2 tissue culture flask
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐027)
  • 0.025% (w/v) trypsin/EDTA (see recipe)
  • 1× embryoid body differentiation medium (1× EBDM; see recipe)
  • Pregnant BALB/c mice time‐mated to embryonic day 12.5
  • 70% (v/v) ethanol
  • Hank's Balanced Salt Solution (HBSS; Invitrogen) containing 1× antibiotic‐antimycotic (AA; penicillin/streptomycin/amphotericin B; obtain 100× stock from Invitrogen)
  • 50 mg/ml dispase (Sigma) in HBSS
  • Coculture substratum (see recipe) in 3.5‐cm diameter petri dishes
  • PicoPure RNA Isolation Kit (Arcturus)
  • Appropriate primers for phenotyping by RT‐PCR (see recipe)
  • 4% (w/v) paraformaldehyde in PBS
  • Rat tail collagen gel, neutralized ( protocol 2)
  • 50%, 70%, 90%, 95%, and 100% (v/v) ethanol
  • Xylene
  • Paraffin
  • Appropriate probes for phenotyping by immunohistochemistry: e.g., anti‐pro‐SP‐C antibody (Chemicon, cat. no. AB3786) or anti‐TTF‐1 antibody (NovoCastra, http://www.vision‐bio.com, cat. no. NCL‐TTF‐1)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • 10‐cm diameter bacteriological‐grade petri dishes (BD Falcon)
  • Dissecting instruments (soaked in 70% ethanol and rinsed in sterile PBS prior to use):
    • Dissecting scissors, sharp
    • Dissecting forceps
    • Micro‐dissecting scissors (Fine Science Tools)
    • Fine‐tipped dissecting forceps (Fine Science Tools)
  • Dissecting microscope
  • Glass embryo dish (Canemco & Marivac; http://www.canemco.com)
  • 125‐µm tungsten needles and needle holders (Fine Science Tools)
  • Finely drawn glass Pasteur pipets and rubber bulb
  • Embedding cassette
  • Automated embedding machine
  • Microtome
  • Additional reagents and equipment for cell culture techniques including counting cells (Phelan, ), euthanasia of the mouse (Donovan and Brown, ), reverse transcription–polymerase chain reaction (RT‐PCR; Beverley, ), preparation for immunohistochemistry including paraffin embedding (Zeller, ), and immunohistochemistry (Watkins, )
NOTE: All incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Support Protocol 1: Preparation of Neutralized Rat‐Tail Collagen Gel

  Materials
  • Rat tail collagen (Sigma, cat. no. C‐8897)
  • 0.1% (v/v) acetic acid
  • Medium 199 (Sigma)
  • 0.35 M NaOH (Sigma)
  • Centrifuge with Beckman JA‐14 rotor (or equivalent)

Alternate Protocol 1: Indirect Coculture of Murine EBs and Murine Embryonic Distal Lung Mesenchyme

  • 24‐well tissue culture plates
  • 0.02‐µm Anapore membrane tissue culture inserts for 24‐well plates (Whatman)
NOTE: All incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Derivation of Early Distal Lung Epithelial Progenitors from Murine ES Cells

  Materials
  • Undifferentiated murine ES cell culture (feeder‐free) in gelatinized (see recipe) 25‐cm2 tissue culture flask
  • Definitive Endoderm‐Inducing Medium (DEIM; see recipe) with and without activin A
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐027)
  • Small Airway Basal Medium (SABM; Cambrex)
  • 10‐cm‐diameter bacteriological petri dish
  • 6‐well tissue culture plate or 75‐cm2 tissue culture flask, gelatinized (see recipe)
  • Additional reagents and equipment for forming EBs ( protocol 1, steps 1 to 7)
NOTE: All incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Derivation of Distal Respiratory Epithelial Cells from Human ES Cells

  Materials
  • Undifferentiated human ES cell (hESC) colonies on mitotically inactivated murine embryonic fibroblast feeder cells in 6‐well plates
  • Medium for maintaining undifferentiated hESC
  • 1 mg/ml collagenase IV (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐027)
  • hEB medium (see recipe)
  • Dif°hES medium (see recipe)
  • Small Airway Growth Medium (SAGM; Cambrex)
  • Felt‐tip pen
  • Inverted microscope
  • Glass Pasteur pipet with tip broken, autoclaved 15‐ml centrifuge tubes
  • Centrifuge
  • 90‐mm cell adherence–resistant Nalgene polymethylpentene petri dishes (Nalge Nunc)
  • 6‐well tissue culture–treated plates with high‐grade polystyrene culture surfaces (Orange Scientific; http:// www.orangesci.com)
NOTE: All incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
   Alcorn, J.L., Smith, M.E., Smith, J.F., Margraf, L.R., and Mendelson, C.R. 1997. Primary cell culture of human type II pneumonocytes: Maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. Am. J. Respir. Cell. Mol. Biol. 17:672‐682.
   Ali, N.N., Edgar, A.J., and Samdikuchaksaraei, A. 2002. Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng. 8:541‐549.
   Beverley, S. M. 2001. Enzymatic amplification of RNA by PCR (RT‐PCR). Curr. Protoc. Mol. Biol. 56:15.5.1‐15.5.6.
   Bogue, C.W., Jacobs, H.C., Dynia, D.W., Wilson, C.M., and Gross, I. 1996. Retinoic acid increases surfactant protein mRNA in fetal rat lung in culture. Am. J. Physiol. 271:L862‐L868.
   D'Amour, K.A., Agulnick, A.D., Eliazer, S., Kelly, O.G., Kroon, E., and Baetge, E.E. 2005. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23:1534‐1541.
   D'Amour, K.A., Bang, A.G., Eliazer, S., Kelly, O.G., Agulnick, A.D., Smart, N.G., Moorman, M.A., Kroon, E., Carpenter, M.K., and Baetge, E.E. 2006. Production of pancreatic hormone‐expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24:1392‐1401.
   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. 1985. The in vitro development of blastocyst‐derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87:27‐45.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Edwards, L.A., Read, L.C., Nishio, S.J., Weir, A.J., Hull, W., Barry, S., Styne, D., Whitsett, J.A., Tarantal, A.F., and George‐Nascimento, C. 1995. Comparison of the distinct effects of epidermal growth factor and betamethasone on the morphogenesis of the gas exchange region and differentiation of alveolar type II cells in lungs of fetal rhesus monkeys. J. Pharmacol. Exp. Ther. 274:1025‐1032.
   Fair, J.H., Cairns, B.A., Lapaglia, M., Wang, J., Meyer, A.A., Kim, H., Hatada, S., Smithies, O., and Pevny, L. 2003. Induction of hepatic differentiation in embryonic stem cells by co‐culture with embryonic cardiac mesoderm. Surgery 134:189‐196.
   Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S.I., and Sasai, Y. 2000. Induction of midbrain dopaminergic neurons from ES cells by stromal cell‐derived inducing activity. Neuron 28:31‐40.
   Keller, G.M. 1995. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell. Biol. 7:862‐869.
   Kubo, A., Shinozaki, K., Shannon, J.M., Kouskoff, V., Kennedy, M., Woo, S., Fehling, H.J., and Keller, G. 2004. Development of definitive endoderm from embryonic stem cells in culture. Development 131:1651‐1662.
   Leahy, A., Xiong, J.W., Kuhnert, F., and Stuhlmann, H. 1999. Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J. Exp. Zool. 284:67‐81.
   Lowe, L.A., Yamada, S., and Kuehn, M.R. 2001. Genetic dissection of nodal function in patterning the mouse embryo. Development 128:1831‐1843.
   Madsen, J., Tornoe, I., Nielsen, O., Koch, C., Steinhilber, W., and Holmskov, U. 2003. Expression and localization of lung surfactant protein A in human tissues. Am. J. Respir. Cell. Mol. Biol. 29:591‐597.
   Mateizel, I., De Temmerman, N., Ullmann, U., Cauffman, G., Sermon, K., Van de Velde, H., De Rycke, M., Degreef, E., Devroey, P., Liebaers, I., and Van Steirteghem, A. 2006. Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum. Reprod. 21:503‐511.
   Nakano, T., Kodama, H., and Honjo, T. 1996. In vitro development of primitive and definitive erythrocytes from different precursors. Science 272:722‐724.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Phelps, D.S., and Floros, J. 1991. Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization. Exp. Lung Res. 17:985‐995.
   Plopper, C.G., St. George, J.A., Read, L.C., Nishio, S.J., Weir, A.J., Edwards, L., Tarantal, A.F., Pinkerton, K.E., Merritt, T.A., Whitsett, J.A., et al. 1992. Acceleration of alveolar type II cell differentiation in fetal rhesus monkey lung by administration of EGF. Am. J. Physiol. 262:L313‐L321.
   Pryhuber, G.S. 1998. Regulation and function of pulmonary surfactant protein B. Mol. Genet. Metab. 64:217‐228.
   Rippon, H.J., Ali, N.N., Polak, J.M., and Bishop, A.E. 2004. Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells. Cloning Stem Cells 6:49‐56.
   Rippon, H.J., Polak, J.M., Qin, M., and Bishop, A.E. 2006. Derivation of distal lung epithelial progenitors from murine embryonic stem cells using a novel three‐step differentiation protocol. Stem Cells 24:1389‐1398.
   Samadikuchaksaraei, A., Cohen, S., Isaac, K., Rippon, H.J., Polak, J.M., Bielby, R.C., and Bishop, A.E. 2006. Derivation of distal airway epithelium from human embryonic stem cells. Tissue Eng. 12:867‐875.
   Shannon, J.M. 1994. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev. Biol. 166:600‐14.
   Strayer, M.S., Guttentag, S.H., and Ballard, P.L. 1998. Targeting type II and Clara cells for adenovirus‐mediated gene transfer using the surfactant protein B promoter. Am. J. Respir. Cell. Mol. Biol. 18:1‐11.
   Sui, Y., Clarke, T., and Khillan, J.S. 2003. Limb bud progenitor cells induce differentiation of pluripotent embryonic stem cells into chondrogenic lineage. Differentiation 71:578‐585.
   Sun, Y., Li, H., Yang, H., Rao, M.S., and Zhan, M. 2006. Mechanisms controlling embryonic stem cell self‐renewal and differentiation. Crit. Rev. Eukaryot. Gene Expr. 16:211‐231.
   Tada, S., Era, T., Furusawa, C., Sakurai, H., Nishikawa, S., Kinoshita, M., Nakao, K., Chiba, T., and Nishikawa, S. 2005. Characterization of mesendoderm: A diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132:4363‐4374.
   Van Vranken, B.E., Romanska, H.M., Polak, J.M., Rippon, H.J., Shannon, J.M., and Bishop, A.E. 2005. Coculture of embryonic stem cells with pulmonary mesenchyme: A microenvironment that promotes differentiation of pulmonary epithelium. Tissue Eng. 11:1177‐1187.
   Voorhout, W.F., Weaver, T.E., Haagsman, H.P., Geuze, H.J., and Van Golde, L.M. 1993. Biosynthetic routing of pulmonary surfactant proteins in alveolar type II cells. Microsc. Res. Tech. 26:366‐373.
   Watkins, S. 1989. Immunohistochemistry. Curr. Protoc. Mol. Biol. 7:14.6.1‐14.6.13.
   Xu, X., McCormick‐Shannon, K., Voelker, D.R., and Mason, R.J. 1998. KGF increases SP‐A and SP‐D mRNA levels and secretion in cultured rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 18:168‐178.
   Yasunaga, M., Tada, S., Torikai‐Nishikawa, S., Nakano, Y., Okada, M., Jakt, L.M., Nishikawa, S., Chiba, T., Era, T., and Nishikawa, S. 2005. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat. Biotechnol. 23:1542‐1550.
   Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1‐14.1.8.
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