C99-01 Induced Pluripotent Stem Cell Derived Pulmonary Ionocytes Rescue CFTR Mediated Ion Transport in Cystic Fibrosis Airway Epithelium

Abstract Rationale Pulmonary ionocytes, distinguished by the canonical marker FOXI1, represent a minority cell population in murine and human airways, expressing over 90% of CFTR transcripts. Despite their rarity, their functional relevance in airway ion and fluid transport regulation remains unclear. While the high level of CFTR transcript expression suggests a role for ionocytes in CF secretory cells represent the largest population of CFTR expressing cells within the airway and there is on going discussion of the utility of either cell type as a target for gene editing or cellular transplantation for future CF therapeutics. To address this gap, we successfully differentiated human iPSCs into airway epithelium, generating both major cell types and pulmonary ionocytes transcriptomically akin to primary counterparts. By modulating FOXI1 expression ionocyte presence can be increased or eliminated from the airway epithelium allowing for titratable studies of the impact of pulmonary ionocytes on WT and CF airways Methods FOXI1-mutant and WT syngeneic iPSCs were generated using a Cas9/CRISPR system. Additionally, we produced syngeneic clones of dox-FOXI1-OE iPSCs using a PiggyBac transgene construct featuring a DOX-inducible FOXI1-2a-mCherry expression cassette allowing for conditional expression of FOXI1. FOXI1- mutant, Dox-inducible FOXI1-OE, and syngeneic FOXI1-unedited iPSCs were differentiated to airway epithelium cultured at air-liquid interface (ALI) using our previously published directed differentiation protocol. Cultures underwent transcript (RT-PCR), protein (IFA) and physiologic (Isc potential) analysis. Results FOXI1 knockout in iPSC-derived airway epithelium eliminates pulmonary ionocytes, indicated by reduced ASCL3, BSND, and CFTR expression, and absence of FOXI1+/BSND+ cells on immunostaining. FOXI1-KO airway epithelium exhibits MUC5B+ secretory cells, and decreased α-Tubulin+ ciliary cells, and alters the expression of multiple canonical airway markers (increased MUC5B, SCGB1A1, and ASCL1, and decreased TP63 and FOXJ1). FOXI1-KO diminishes epithelial CFTR-dependent Forskolin-induced ion transport. Conditional over expression of FOXI1 in a FOXI1-KO background restores the presence of ionocytes and CFTR-dependent Forskolin-induced ion transport. Introduction of CFTR wild type FOXI1-OE cells into a CFTR F508del/F508del background rescued CFTR-dependent Forskolin-induced ion transport. Conclusions We show that disruption of FOXI1 results in a reduction of CFTR-dependent chloride transport in iPSC-derived airway epithelium, suggesting a potential role of ionocytes in regulating airway epithelial function. Additionally, we show that ionocytes influence cell lineage fate in the respiratory epithelium including the proportion of secretory cells. Introduction of WT CFTR expressing ionocyte into a CF airway epithelial culture restored CFTR mediated Cl_ suggesting a potential role for ionocytes as a therapeutic target This abstract is funded by: CFF, NIH