Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells

J Gene Med. 2004 Aug;6(8):846-56. doi: 10.1002/jgm.570.


Background: Following injury to the airway epithelium, rapid regeneration of a functional epithelium is necessary in order to restore the epithelial barrier integrity. In the perspective of airway gene/cell therapy, we analyzed the capacity of human airway epithelial cells cultured as three-dimensional (3-D) spheroid structures to be efficiently transduced on long term by a pseudotyped lentiviral vector. The capacity of the 3-D spheroid structures to repopulate a denuded tracheal basement membrane and regenerate a well-differentiated airway epithelium was also analyzed.

Methods: An HIV-1-derived VSV-G pseudotyped lentiviral vector encoding the enhanced green fluorescent protein (eGFP) was used. Airway epithelial cells were isolated from mature human fetal tracheas and airway xenografts, cultured as 3-D spheroid structures, and either transduced at multiplicity of infection (MOI) 10 and 100 or assayed in an ex vivo and in vivo model to evaluate their regeneration capacity.

Results: An in vivo repopulation assay in SCID-hu mice with transduced isolated fetal airway epithelial cells shows that lentiviral transduction does not alter the airway reconstitution. Transduction of the 3-D spheroid structures shows that 12% of cells were eGFP-positive for up to 80 days. In ex vivo and in vivo assays (NUDE-hu mice), the 3-D spheroid structures are able to repopulate denuded basement membrane and reconstitute a well-differentiated human airway surface epithelium.

Conclusions: The efficient and long-term lentiviral transduction of 3-D spheroid structures together with their capacity to regenerate a well-differentiated mucociliary epithelium demonstrate the potential relevance of these 3-D structures in human airway gene/cell therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • Cells, Cultured
  • Epithelial Cells / physiology*
  • Epithelium / physiology*
  • Gene Transfer Techniques*
  • Genetic Vectors*
  • Humans
  • Lentivirus / genetics*
  • Mice
  • Mice, Nude
  • Microscopy, Fluorescence
  • Regeneration*
  • Spheroids, Cellular / physiology*
  • Trachea / cytology
  • Trachea / physiology
  • Transduction, Genetic