Key factors for the differentiation of embryonic and induced pluripotent stem cells into respiratory epithelial cells

The ability to ex-vivo produce respiratory epithelial cell types would offer novel cellular therapies targeting acquired or hereditary diseases of the airways and parenchyma of the lung, like cystic fibrosis and surfactant deficiencies. Pluripotent stem cells (PSCs) may represent a suitable cell source, but the efficiency of existing differentiation protocols towards lung epithelial cells is still low. Therefore, we aimed at identifying key factors which drive the differentiation of murine embryonic (ESCs) as well as murine induced pluripotent stem cells (iPSCs) into airway epithelial Clara cells and alveolar epithelial type II (ATII) cells. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is known to be a strong growth factor at least for alveolar epithelial cells. Similar to KGF, glucocorticoids also have a stimulating effect on the maturation of primary lung epithelial cells, and this effect is even potentiated by the simultaneous addition of cAMP-elevating agents. We thus hypothesized that the addition of KGF either alone or together with the three-factor combination dexamethasone, 8-bromoadenosine-cAMP and isobutylmethylxanthine (DCI) supports the pulmonary differentiation of ESCs and iPSCs. Indeed, we identified a synergistic effect of KGF and DCI on the differentiation into ATII cells with a surprising need for an early KGF supplementation already starting at day 0 or 5 of differentiation; suggesting a so far unknown effect of KGF in the earliest phase of differentiation. This synergistic stimulating effect resulted in significantly increased mRNA expression of the ATII cell-specific markers surfactant protein (SP-)C and SP-B in ESC as well as iPSC differentiation cultures. In contrast to ATII cells, expression of the Clara cell-specific marker, Clara cell secretory protein (CCSP), was most induced following DCI treatment without KGF. Furthermore, electron microscopy measurements confirmed the phenotype of the PSC-derived respiratory epithelial cells and by means of iPSC clones with reporter gene expression we were able to directly visualize and monitor the successful differentiation of iPSCs into ATII cells and Clara cells. As a first step towards clinical application, we are currently investigating whether KGF and DCI have similar effects on the differentiation of human PSCs as well.

In conclusion, we have identified key factors for the respiratory differentiation of ESCs and iPSCs, which will facilitate more robust and efficient generation of PSC-derived respiratory epithelial cells for future basic research and potential therapeutic application. Furthermore, respiratory reporter gene expression enabled the first reliable proof for the generation of iPSC-derived Clara cells and ATII cells.

*These authors contributed equally