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Differentiation of Induced Pluripotent Stem Cells iPS into Human Bronchial Epithelium Using a Three-Dimensional Organoid System
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A3830 - Differentiation of Induced Pluripotent Stem Cells iPS into Human Bronchial Epithelium Using a Three-Dimensional Organoid System
Author Block: E. Ahmed1, S. Assou1, M. Fieldes1, A. Petit2, I. Vachier3, P. Chanez4, J. De Vos1, A. Bourdin5; 1Institute for Regenerative Medicine and Biotherapy INSERM 1183, Montpellier, France, 2CHU Arnaud de Villeneuve, Montpellier, France, 3CHRU Montpellier, Montpellier, France, 4Aix Marseille Université, Marseille, France, 5CHU Hôpital Arnaud de Villeneuve, MONTPELLIER Cedex 5, France.
RATIONALE Developing new models is mandatory for respiratory research as critical differences exist between human and rodent airway epithelium. Three-dimensional 3D organoid cultures have led to new physiologically complex in vitro models to study human development and disease. Recent reports show that it is possible to reconstitute in vitro a complex and functional airway epithelium displaying all the characteristic features described in vivo from hiPS.
Our aim is to establish a robust and reproducible step-wise differentiation protocol of hiPS differentiation towards human bronchial organoids HBOs.
METHODS Manipulation of developmental signaling pathways such as canonical TGF-beta pathway, Wnt and BMP signaling allows the generation of NKX2.1 bronchial progenitors. Efficiency was evaluated at each step by flow cytometry and immunofluorescence. CXCR4 expression was used as a marker of definitive endoderm, and NKX2.1 of bronchial progenitors.
RESULTS Activin-CHIR99021 protocol lead to the highest CXCR4 induction rate of Definitive Endoderm DE, more than 90% 4 cell iPS lines. Initial medium supplementation with Y27632 was mandatory, at least in part to prevent dissociation-induced apoptosis. IPS-derived NKX2.1+ cell population were then plated into Geltrex matrix and formed well-organized HBOs. Differentiation protocol leaded to a high induction of NKX2.1 progenitors mean value= 78% n=6.
HBOs exhibited distal airway-like epithelium with Keratin 5 basal +cells, FOXJ1 + immature ciliated cells, and CCSP+ club cells that could be maintained forty days in culture.
CONCLUSION High efficiency of the DE and bronchial progenitors NKX2.1 differentiation steps is mandatory to consistently generate bronchial epithelium. 3D human bronchial organoids represent a great tool that recapitulate the lung development in vitro, and is a promising tool to recapitulate lung diseases in vitro.
Author Block: E. Ahmed1, S. Assou1, M. Fieldes1, A. Petit2, I. Vachier3, P. Chanez4, J. De Vos1, A. Bourdin5; 1Institute for Regenerative Medicine and Biotherapy INSERM 1183, Montpellier, France, 2CHU Arnaud de Villeneuve, Montpellier, France, 3CHRU Montpellier, Montpellier, France, 4Aix Marseille Université, Marseille, France, 5CHU Hôpital Arnaud de Villeneuve, MONTPELLIER Cedex 5, France.
RATIONALE Developing new models is mandatory for respiratory research as critical differences exist between human and rodent airway epithelium. Three-dimensional 3D organoid cultures have led to new physiologically complex in vitro models to study human development and disease. Recent reports show that it is possible to reconstitute in vitro a complex and functional airway epithelium displaying all the characteristic features described in vivo from hiPS.
Our aim is to establish a robust and reproducible step-wise differentiation protocol of hiPS differentiation towards human bronchial organoids HBOs.
METHODS Manipulation of developmental signaling pathways such as canonical TGF-beta pathway, Wnt and BMP signaling allows the generation of NKX2.1 bronchial progenitors. Efficiency was evaluated at each step by flow cytometry and immunofluorescence. CXCR4 expression was used as a marker of definitive endoderm, and NKX2.1 of bronchial progenitors.
RESULTS Activin-CHIR99021 protocol lead to the highest CXCR4 induction rate of Definitive Endoderm DE, more than 90% 4 cell iPS lines. Initial medium supplementation with Y27632 was mandatory, at least in part to prevent dissociation-induced apoptosis. IPS-derived NKX2.1+ cell population were then plated into Geltrex matrix and formed well-organized HBOs. Differentiation protocol leaded to a high induction of NKX2.1 progenitors mean value= 78% n=6.
HBOs exhibited distal airway-like epithelium with Keratin 5 basal +cells, FOXJ1 + immature ciliated cells, and CCSP+ club cells that could be maintained forty days in culture.
CONCLUSION High efficiency of the DE and bronchial progenitors NKX2.1 differentiation steps is mandatory to consistently generate bronchial epithelium. 3D human bronchial organoids represent a great tool that recapitulate the lung development in vitro, and is a promising tool to recapitulate lung diseases in vitro.
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