Generation of multiciliated cells in functional airway epithelium from human iPSC (1094.5)

Abstract

Despite a significant improvement in the availability of therapeutic options to treat lung diseases, pulmonary disease still remains a major cause of morbidity and mortality around the world. Currently there are limited opportunities to study human lung disease either in vivo and in vitro. Using induced pluripotent stem cells (iPSC) we have generated a reproducible differentiation protocol to make mature post‐mitotic multiciliated cells in a functional airway epithelium.iPSC were generated from human skin biopsies and differentiated via FOXA2+SOX17+ definitive endoderm (>90% efficiency) to FOXA2+NKx2.1+ anterior foregut endoderm, FOXA2+NKx2.1+SOX2+ (~50% efficiency) pulmonary endoderm and then matured in an air liquid interface. Robust multiciliogenesis occurred when Notch signaling was inhibited and was confirmed by; i) the assembly of multiple pericentrin stained centrioles at the apical surface, ii) expression of transcription factor FOXJ1 and iii) presence of multiple acetylated tubulin labeled cilia projections in individual cells. The presence of NKx2.1+CC10+ Clara cells, MUC5A/C+ goblet cells and FOXA2+p63+ basal cells was also confirmed showing we are generating a complete polarized epithelial cell layer comprised of all relevant cell types. Functional cAMP activated and CFTRinh‐172 sensitive CFTR currents were recorded in isolated epithelial cells by whole cell patch clamp technique. Furthermore, we have corrected the deltaF508 mutation in the CFTR gene (>80% of all cases of CF) using a combination of CRISPR‐Cas9 endonuclease‐mediated genome editing and piggyBac transposase technologies, in the CF patient‐derived iPSC.The generation of mature multiciliated cells in a human iPSC differentiated respiratory epithelium and the ability to correct disease causing mutations provides a significant advancement toward modeling a number of human respiratory diseases in vitro.Grant Funding Source: Supported in part by CIRM and the Berger Foundation