Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

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

Multiciliated Cells in Animals

“…In particular, the identification of the regulatory cascade controlling the specification and differentiation of MCCs in vertebrates opens important avenues in terms of both fundamental knowledge and biomedical applications. The ability to differentiate human MCCs in vitro from induced pluripotent stem cells constitutes a promising therapeutic approach for treating respiratory diseases known to affect the differentiation and function of MCCs, such as primary ciliary dyskinesia, cystic fibrosis, and severe asthma (Thomas et al 2010;Firth et al 2014;Hoegger et al 2014).…”

BMP signalling controls the construction of vertebrate mucociliary epithelia

“…Recent studies have developed protocols to generate in vitro airway epithelial cells from human pluripotent stem cells that include an early inhibition of the BMP pathway followed by its activation in order to push the definitive endoderm to differentiate into ventral anterior foregut, before the induction of lung progenitor specification (Firth et al, 2014;Huang et al, 2015Huang et al, , 2014. However, these reports did not explore the role of BMP signalling at later steps, when airway progenitors give rise to fully differentiated airway MCE.…”

Airway tissue engineering for congenital laryngotracheal disease

“…iPSCs derived from human dermal fibroblasts differentiate to form both proximal and distal airway epithelial cell types under culture conditions which mimic developmental processes [51][52][53][54][55].…”