Primary Ciliary Dyskinesia Patient-Specific hiPSC-Derived Airway Epithelium in Air-Liquid Interface Culture Recapitulates Disease Specific Phenotypes In Vitro

Abstract

Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in DNAH5 and NME5, respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.

Structure-Function Relationships Of Mucociliary Clearance In Human Airways

“…The systems evaluated included mature and developing mouse trachea, human airway epithelial cultures derived from human induced pluripotent stem cells, and primary human airway epithelial cultures subjected to asthma-like inflammatory conditions (interleukin-13 (IL-13) treated) or differentiated under mechanical stimulation (Organ-on-Chip models). We found that the pluripotent stem cells-derived airway epithelial tissue differentiated in PC at ALI (iALIs) as described earlier 53 greatly increased cilia coverage from day 14 and reached nearly human-like levels at day 35 at ALI; however, CPB remained much lower than expected from the extent of cilia coverage, suggesting immature ciliated cell function and organization ( Fig. 6b , purple triangles).…”

“…Differentiation of human iPSCs (hiPSCs) towards respiratory epithelium was performed as described previously 53 . Briefly, hiPSCs from N=1 donor were differentiated to definitive endoderm by using the STEMdiff ™ Definitive Endoderm Kit (STEMCELL Tech., Vancouver, BC, Canada).…”

Ciliary Ultrastructure Assessed by Transmission Electron Microscopy in Adults with Bronchiectasis and Suspected Primary Ciliary Dyskinesia but Inconclusive Genotype

“…In our patient, we performed additional familial segregation analysis of the homozygous variant in NEK10, including parents as well as affected and unaffected siblings, thus suggesting disease causation. Likewise, in the meantime, we produced additional evidence for the relevance of the detected likely pathogenic homozygous variant in NME5 [34], which is beyond the scope of the present work. Lastly, our findings confirm that the PCD Detect software is an easy-to-apply and useful tool, in particular when analyzing faintly contrasted ciliary components like the IDA [17].…”

A Fast Scoring of Human Primary Respiratory Epithelia Grown at Air–Liquid Interface (ALI) to Assess Epithelial Morphology in Research and Personalized Medicine Settings

“…Respiratory diseases can be studied using primary epithelia grown at air-liquid interface (ALI) from patients' biomaterials [1]. ALI epithelia have been described using nasal brushings [2][3][4][5], nasal biopsies [6], bronchial biosamples [5,7], explanted lung tissue [1,8,9], nasal polyps [10,11] or stem cells [12][13][14] as sources to regenerate primary epithelia. To generate primary epithelia, the airway epithelium continuity of the patient's biomaterial needs to be destroyed and the presence of primary airway progenitor cells in the biomaterial needs to be maintained to achieve cell expansion.…”

“…Primary ALI epithelia have been used to characterize human diseases such as the monogenic diseases primary cilia dyskinesia [12] and cystic fibrosis [3,5,12] as well as complex diseases such as asthma [7,17] and chronic obstructive pulmonary disease [8,18]. ALI epithelia are widely employed to test drugs [19][20][21] and to understand environmental influences [22,23], host defense [24,25] or genetic variants [3,10].…”