Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells.
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.
INSERM UMRS 514, IFR 53, CHU Maison-Blanche, 45 rue Cognacq-Jay, Reims, France.
Nicolas
N
Castillon
Aurélie
A
Avril-Delplanque
Christelle
C
Coraux
Christophe
C
Delenda
Bruno
B
Péault
Olivier
O
Danos
Edith
E
Puchelle
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology*; D004848:Epithelium / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D012038:Regeneration; D018874:Spheroids, Cellular / Q000502:physiology*; D014132:Trachea / Q000166:cytology / Q000502:physiology; D014161:Transduction, Genetic