Generation of Clara cells from murine pluripotent stem cells – a new tool to explore airway epithelial regeneration

Airway epithelial cell production in vitro offers new options to treat airway diseases, including genetic disorders like cystic fibrosis. Pluripotent stem cells (PSCs) (embryonic (ESCs) or induced pluripotent stem cells (iPSCs)) represent a suitable exogenous cell source for cell replacement strategies. Aiming at the long-term restoration of functional airway epithelium, epithelial progenitor/stem cells will be required, e.g. Clara cells. Clara cells are able to regenerate the airway epithelium following injury. With the aim to establish a mouse model of long-term airway epithelial regeneration, we aimed at the in vitro generation of Clara cells from murine PSCs. Using iPSCs established from two different Clara cell reporter mouse strains enabled identification of generated Clara cells.

iPSCs from CCSP-rtTA2 s-M2/GFP-tetO7-lacZ mice as well as ESCs were differentiated towards Clara cells using a serum-free monolayer (ML) protocol. The medium was supplemented with dexamethasone, 8-Bromo-cAMP and isobutylmethylxanthine (DCI), with or without keratinocyte growth factor (KGF). Specific marker expression was measured by qRT-PCR. iPSC-derived lacZpos Clara cells were visualized via X-gal staining and were further analyzed by electron microscopy. Pre-differentiated iPSCs were injected under the kidney capsule of immunodeficient mice and analyzed two weeks later. Furthermore, we established additional iPSC clones from CCSP-2A/YFP-2A/iCre knock-in mice.

We have identified the factor combination DCI as an import inducer of the Clara cell marker CCSP in differentiation cultures of murine PSCs. The CCSP-driven expression of lacZ enabled the monitoring of iPSC-derived Clara cells and the confirmation of the Clara cell phenotype in isolated lacZpos areas by enhanced CCSP mRNA expression and a Clara cell typical ultrastructure. Moreover, the iPSC-derived lacZpos cells formed epithelial-like structures in vivo with similarities to lacZpos airways of the Clara cell reporter mice. The recently established iPSC clones from CCSP-2A/YFP-2A/iCre knock-in mice were already successfully differentiated into YFPpos cells using the DCI supplemented ML protocol.