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  • Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells.

Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells.

Nature protocols (2015-02-06)
Amy P Wong, Stephanie Chin, Sunny Xia, Jodi Garner, Christine E Bear, Janet Rossant
ABSTRACT

Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in <5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Gelatin from porcine skin, powder, gel strength ~300 g Bloom, Type A, BioReagent, suitable for electrophoresis, suitable for cell culture
Sigma-Aldrich
3-Isobutyl-1-methylxanthine, ≥99% (HPLC), powder
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Amiloride hydrochloride hydrate, ≥98% (HPLC), powder
Sigma-Aldrich
Fibroblast Growth Factor-18 human, >95% (SDS-PAGE and HPLC), recombinant, expressed in E. coli, lyophilized powder
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Bovine Serum Albumin, lyophilized powder, low endotoxin, BioReagent, suitable for cell culture, ≥98% (agarose gel electrophoresis)
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Genistein, synthetic, ≥98% (HPLC), powder
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HEPES, ≥99.5% (titration)
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Collagen from human placenta, Bornstein and Traub Type IV, powder
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Mitomycin C from Streptomyces caespitosus, powder, contains NaCl as solubilizer
Sigma-Aldrich
Forskolin, from Coleus forskohlii, ≥98% (HPLC), powder