Investigating pulmonary ionocyte function and development using iPSCs

Project Summary/Abstract Respiratory diseases often remain inadequately treated due to a limited understanding of the cellular composition and developmental programs within the lung. Recent single-cell studies have identified novel cell types and progenitor cells in the human airway, revealing potential new therapeutic targets. A particularly intriguing discovery is the pulmonary ionocyte, a rare cell type marked by the expression of FOXI1 and ASCL3 and high levels of the cystic fibrosis transmembrane conductance regulator (CFTR), the gene implicated in cystic fibrosis (CF). These cells, which possess long cytoplasmic projections and interact with other airway cell types, play a debated role in airway function across species. While their involvement in airway surface liquid viscosity and ion transport has been observed in mice and ferrets, such roles remain unconfirmed in human in vitro models. This project aims to deepen our understanding of pulmonary ionocytes using human iPSC-derived airway epithelium. Preliminary data show that FOXI1 deletion (KO) in these models alters epithelial composition, increases secretory cells, and decreases ciliated cells, with a concomitant reduction in CFTR-mediated ion transport. Furthermore, FOXI1 overexpression (OE) can reverse these defects, and the introduction of wild-type CFTR ionocytes into F508del homozygous backgrounds restores chloride transport. Additionally, our findings highlight a temporal role for NOTCH signaling in ionocyte development, where combined inhibition of NOTCH and activation of Sonic Hedgehog (SHH) enhances ionocyte numbers. Our research has three primary aims: 1) Elucidate Ionocyte Differentiation Pathways: We will manipulate SHH and NOTCH pathways to increase ionocyte numbers, investigate the mechanisms through single-cell RNA sequencing, and perform chemical screens to identify additional signaling pathways and surface markers for ionocyte enrichment. 2) Investigate Ionocyte Function in Airway Ion/Fluid Transport: We will use FOXI1-KO and FOXI1-OE iPSC lines to assess ionocyte impacts on airway ion/fluid transport and mucociliary clearance. We aim to determine whether FOXI1 overexpression can rescue deficiencies in FOXI1-KO ionocytes and if wild-type ionocytes can restore ion transport in CF airways. 3) Explore Ionocyte Interactions with other airway cell types: Through single-cell RNA sequencing, fluorescent reporters, and high-resolution live-microscopy, we will examine how ionocytes interact with neighboring airway cells and track ionocyte emergence during differentiation. Our project seeks to provide insights into the regulatory mechanisms governing ionocyte differentiation and function, which could pave the way for new therapeutic strategies in treating CF and other airway diseases.