Tissue Differentiation of ESC into Lung Cells and Functional Validation

  • Ena Ray Banerjee


The pulmonary system is composed of a variety of epithelial cell populations residing in distinct anatomical locations. Of these, the alveolar epithelial gas exchange surface consists of two cell types, the type I and type II pneumocytes, also known as alveolar epithelial type I and type II (AEI and AEII) cells, that comprise ~95 % and 5 %, respectively, of the alveolar lining area (Chen et al. 2004). AEI cells, important in the regulation of alveolar fluid balance (Dahlin et al. 2004), are branched cells with cytoplasm extremely attenuated for gas exchange (Weibel 1984). AEII cells are cuboidal cells situated between AEI cells and contain characteristic lamellar bodies and apical microvilli (Weibel 1984). Functions of AEII cells include the secretion and reuptake of pulmonary surfactant (Fehrenbach 2001), regulation of alveolar fluid, and synthesis of immunomodulatory proteins (e.g., surfactant protein (SP)-A, SP-D) important for host defense (Matthay et al. 2002). The non-ciliated columnar Clara cells (Evans et al. 1978) constitute the majority of the bronchiolar and terminal bronchiolar epithelium. Clara cells actively divide and differentiate to form ciliated cells, secrete glycosaminoglycans that are major component of the extracellular matrix (ECM), and metabolize airborne toxins by cytochrome P-450 enzymes present in their smooth endoplasmic reticulum (Bishop 2004).


Idiopathic Pulmonary Fibrosis Embryoid Body Clara Cell Pluripotent Marker Embryonic Stem Cell Medium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Alveolar epithelial type I


Alveolar epithelial type II




Bronchoalveolar lavage


Bronchial epithelial growth medium


Burst-forming unit-erythroid


CREB-binding protein


Clara cell-specific protein-10


Colony-forming unit-erythroid


Colony-forming unit-granulocyte


Colony-forming unit-granulocyte/erythroid/macrophage/megakar-yocyte


Colony-forming unit-granulocyte/macrophage


Colony-forming unit-macrophage


Embryoid body


Extracellular matrix


Fluorescence-activated cell sorting


Fibroblast growth factor


Human embryonic stem




Idiopathic pulmonary fibrosis


Lymphoid enhancer factor-1


Quantitative real-time PCR


Small airway growth medium


Surfactant protein


T cell factor


Transforming growth factor


Vascular endothelial growth factor



We thank Angelique Nelson, Marilyn Nourse, and Carol B. Ware for technical assistance in the culture of hES cells and Bobbie Schneider for technical assistance with the transmission electron microscopy.

Author Contributions

Conceived and designed the experiments: ERB, WRH. Performed the experiments: ERB. Analyzed the data: ERB, WRH. Contributed reagents/materials/analysis tools: MAL, TP, MK, CEM. Wrote the paper: ERB, WRH.

Competing Interests

The authors have declared that no competing interests exist. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials.


Supported by National Institutes of Health grant R01 HL73722. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


  1. Ali NN, Edgar AJ, Samadikuchaksaraei A, Timson CM, Romanska HM et al (2002) Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng 8:541–550PubMedCrossRefGoogle Scholar
  2. Beers MF, Morrisey E (2011) The three R’s of lung health and disease: repair, remodeling, and regeneration. J Clin Invest 121:2065–2073PubMedCrossRefPubMedCentralGoogle Scholar
  3. Bishop AE (2004) Pulmonary epithelial stem cells. Cell Prolif 37:89–96PubMedCrossRefGoogle Scholar
  4. Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL et al (1995) Defective lymphoid development in mice lacking expression of the common cytokine receptor g chain. Immunity 2:223–238PubMedCrossRefGoogle Scholar
  5. Chen Z, Jin N, Narasaraju T, Chen J, McFarland LR et al (2004) Identification of two novel markers for alveolar epithelial type I and II cells. Biochem Biophys Res Commun 319:774–780PubMedCrossRefGoogle Scholar
  6. Chistiakov D (2010) Endogenous and exogenous stem cells: a role in lung repair and use in airway tissue engineering and transplantation. J Biomed Sci 17:92–100PubMedCrossRefPubMedCentralGoogle Scholar
  7. Cho JY, Miller M, Baek KJ, Han JW, Nayar J et al (2004) Inhibition of airway remodeling in IL-5-deficient mice. J Clin Invest 113:551–560PubMedCrossRefPubMedCentralGoogle Scholar
  8. Dahlin K, Mager EM, Allen L, Tigue Z, Goodglick L et al (2004) Identification of genes differentially expressed in rat alveolar type I cells. Am J Respir Cell Mol Biol 31:309–316PubMedCrossRefGoogle Scholar
  9. Emami KH, Nguyen C, Ma H, Kim D-H, Jeong K-W et al (2004) A small molecule inhibitor of b-catenin/CBP-binding protein transcription [corrected]. Proc Natl Acad Sci 101:12682–12687PubMedCrossRefPubMedCentralGoogle Scholar
  10. Evans MJ, Cabral-Anderson LJ, Freeman G (1978) Role of the Clara cell in renewal of the bronchiolar epithelium. Lab Invest 38:648–653PubMedGoogle Scholar
  11. Fehrenbach H (2001) Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2:33–46PubMedCrossRefPubMedCentralGoogle Scholar
  12. German D, Blyszczuk P, Valaperti A, Kania G, Dirnhofer S et al (2009) Prominin-1/CD133+ lung epithelial progenitors protect from bleomycin-induced pulmonary fibrosis. Am J Respir Crit Care Med 179:939–949CrossRefGoogle Scholar
  13. Hecht A, Vleminckx K, Stemmler MP, Van Roy F, Kemler R (2000) The p300/CBP acetyltransferases function as transcriptional coactivators of b-catenin in vertebrates. EMBO J 19:1839–1850PubMedCrossRefPubMedCentralGoogle Scholar
  14. Henderson WR Jr, Chi EY, Ye X, Nguyen C, Tien Y et al (2010) Inhibition of Wnt/b-catenin/CBP signaling reverses pulmonary fibrosis. Proc Natl Acad Sci U S A 107:14309–14314PubMedCrossRefPubMedCentralGoogle Scholar
  15. Hirabayashi Y, Itoh Y, Tabata H, Nakajima K, Akiyama T et al (2004) The Wnt/b-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131:2791–2801PubMedCrossRefGoogle Scholar
  16. Hochedlinger K, Yamada Y, Beard C, Jaenisch R (2005) Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121:465–477PubMedCrossRefGoogle Scholar
  17. Kasper M, Haroske G (1996) Alterations in the alveolar epithelium after injury leading to pulmonary fibrosis. Histol Histopathol 11:463–483PubMedGoogle Scholar
  18. Kolla V, Gonzales LW, Gonzales J, Wang P, Angampalli S et al (2007) Thyroid transcription factor in differentiating type II cells: regulation, isoforms, and target genes. Am J Respir Cell Mol Biol 36:213–225PubMedCrossRefPubMedCentralGoogle Scholar
  19. Kuwano K, Hagimoto N, Hara N (2001) Molecular mechanisms of pulmonary fibrosis and current treatment. Curr Mol Med 1:551–573PubMedCrossRefGoogle Scholar
  20. Laflamme MA, Gold J, Xu C, Hassanipour M, Rosler E et al (2005) Formation of human myocardium in the rat heart from human embryonic stem cells. Am J Pathol 167:663–671PubMedCrossRefPubMedCentralGoogle Scholar
  21. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA et al (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 25:1015–1024PubMedCrossRefGoogle Scholar
  22. Leblond A, Naud P, Forest V, Gourden C, Sagan C et al (2009) Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury. Hum Gene Ther 20:1329–1343PubMedCrossRefPubMedCentralGoogle Scholar
  23. Lu Y, Okubo T, Rawlins E, Hogan BL (2008) Epithelial progenitor cells of the embryonic lung and the role of microRNAs in their proliferation. Proc Am Thorac Soc 5:300–304PubMedCrossRefPubMedCentralGoogle Scholar
  24. Lukaszewicz AI, McMillan M, Kahn M (2010) Small molecules and stem cells. Potency and lineage commitment: the new quest for the fountain of youth. J Med Chem 53:3439–3453PubMedCrossRefPubMedCentralGoogle Scholar
  25. Ma H, Nguyen C, Lee KS, Kahn M (2005) Differential roles for the coactivators CBP and p300 on TCF/b-catenin-mediated survivin gene expression. Oncogene 24:3619–3631PubMedCrossRefGoogle Scholar
  26. Matthay MA, Folkesson HG, Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82:569–600PubMedGoogle Scholar
  27. McMillan M, Kahn M (2005) Investigating Wnt signaling: a chemogenomic safari. Drug Discov Today 10:1467–1474PubMedCrossRefGoogle Scholar
  28. Miyabayashi K, Maruyama M, Yamada T, Shinoda C, Hounoki H et al (2006) Isoproterenol suppresses cytokine-induced RANTES secretion in human lung epithelial cells through the inhibition of c-jun N-terminal kinase pathway. Biochem Biophys Res Commun 350:753–761PubMedCrossRefGoogle Scholar
  29. Moon RT, Kohn AD, De Ferrari GV, Kaykas A (2004) Wnt and b-catenin signalling: diseases and therapies. Nat Rev Genet 5:691–701PubMedCrossRefGoogle Scholar
  30. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D et al (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379–391PubMedCrossRefGoogle Scholar
  31. Niwa H, Miyazaki J, Smith AG (2000) Quanti-tative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372–376Google Scholar
  32. Otero JJ, Fu W, Kan L, Cuadra AE, Kessler JA (2004) b-catenin signaling is required for neural differentiation of embryonic stem cells. Development 131:3545–3557PubMedCrossRefGoogle Scholar
  33. Rawlins EL, Ostrowski LE, Randell SH, Hogan BL (2007) Lung development and repair: contribution of the ciliated lineage. Proc Natl Acad Sci U S A 104:410–417PubMedCrossRefPubMedCentralGoogle Scholar
  34. Ray Banerjee E, LaFlamme MA, Papayannopoulou T, Kahn M, Murry CE, Henderson WR Jr (2012) Human embryonic stem cells differentiated to lung lineage-specific cells ameliorate pulmonary fibrosis in a xenograft transplant mouse model. PLoS One 7(3):e33165:1–15Google Scholar
  35. Rippon HJ, Ali NN, Polak JM, Bishop AE (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 6:49–56PubMedCrossRefGoogle Scholar
  36. Rippon HJ, Polak JM, Qin M, Bishop AE (2006) Derivation of distal lung epithelial progenitors from murine embryonic stem cells using a novel three-step differentiation protocol. Stem Cells 24:1389–1398PubMedCrossRefGoogle Scholar
  37. Samadikuchaksaraei A, Cohen S, Isaac K, Rippon HJ, Polak JM et al (2006) Derivation of distal airway epithelium from human embryonic stem cells. Tissue Eng 12:867–875PubMedCrossRefGoogle Scholar
  38. Stripp BR, Reynolds SD (2005) Bioengineered lung epithelium: implications for basic and applied studies in lung tissue regeneration. Am J Respir Cell Mol Biol 32:85–86PubMedCrossRefGoogle Scholar
  39. Takemaru KI, Moon RT (2000) The transcriptional coactivator CBP interacts with b-catenin to activate gene expression. J Cell Biol 149:249–254PubMedCrossRefPubMedCentralGoogle Scholar
  40. Teo JL, Ma H, Nguyen C, Lam C, Kahn M (2005) Specific inhibition of CBP/b-catenin interaction rescues defects in neuronal differentiation caused by a presenilin-1 mutation. Proc Natl Acad Sci U S A 102:12171–12176PubMedCrossRefPubMedCentralGoogle Scholar
  41. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147PubMedCrossRefGoogle Scholar
  42. Uhal BD (1997) Cell cycle kinetics in the alveolar epithelium. Am J Physiol 272:L1031–L1045PubMedGoogle Scholar
  43. Wang D, Haviland D, Burns A, Zsigmond E, Wetsel R (2007) A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 104:4449–4454PubMedCrossRefPubMedCentralGoogle Scholar
  44. Warburton D, Perin L, Defilippo R, Bellusci S, Shi W et al (2008) Stem/progenitor cells in lung development, injury repair, and regeneration. Proc Am Thorac Soc 5:703–706PubMedCrossRefPubMedCentralGoogle Scholar
  45. Ware CB, Nelson AM, Blau CA (2005) Controlled-rate freezing of human ES cells. BioTechniques 38:879–3PubMedCrossRefGoogle Scholar
  46. Weibel ER (1984) The pathway for oxygen. Harvard University Press, Cambridge, MA, pp 231–271Google Scholar
  47. Wetsel R, Wang D, Calame D (2011) Therapeutic potential of lung epithelial progenitor cells derived from embryonic and induced pluripotent stem cells. Annu Rev Med 62:95–105PubMedCrossRefGoogle Scholar
  48. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED et al (2005) Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-b1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 166:1321–1332PubMedCrossRefPubMedCentralGoogle Scholar
  49. Witschi H (1991) Role of the epithelium in lung repair. Chest 99:22S–25SPubMedCrossRefGoogle Scholar
  50. Zechner D, Fujita Y, Hulsken J, Muller T, Walther I et al (2003) b-catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev Biol 258:406–418PubMedCrossRefGoogle Scholar
  51. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K et al (2001) Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 33:907–921PubMedCrossRefGoogle Scholar

Copyright information

© Springer India 2014

Authors and Affiliations

  • Ena Ray Banerjee
    • 1
  1. 1.Department of ZoologyUniversity of CalcuttaKolkataIndia

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