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

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.

[1]  J. Nawroth,et al.  Breathing on Chip: Dynamic flow and stretch tune cellular composition and accelerate mucociliary maturation of airway epithelium in vitro , 2021, bioRxiv.

[2]  Romain F. Laine,et al.  TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines , 2022, Nature Methods.

[3]  S. Randell,et al.  A multimodal iPSC platform for cystic fibrosis drug testing , 2021, Nature Communications.

[4]  H. Clevers,et al.  Modelling of primary ciliary dyskinesia using patient‐derived airway organoids , 2021, EMBO reports.

[5]  J. Parkinson,et al.  A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients , 2021, Stem cell reports.

[6]  M. Hagiwara,et al.  Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology , 2021, Science Translational Medicine.

[7]  E. Ziętkiewicz,et al.  Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia , 2021, International journal of molecular sciences.

[8]  M. Odijk,et al.  Measuring barrier function in organ-on-chips with cleanroom-free integration of multiplexable electrodes , 2021, Lab on a chip.

[9]  P. Beales,et al.  Higher throughput drug screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia , 2021, European Respiratory Journal.

[10]  S. Kalies,et al.  High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling , 2021, Stem cells translational medicine.

[11]  U. Martin,et al.  Production and cryopreservation of definitive endoderm from human pluripotent stem cells under defined and scalable culture conditions , 2021, Nature Protocols.

[12]  F. Pessler,et al.  Generation of two human ISG15 knockout iPSC clones using CRISPR/Cas9 editing. , 2020, Stem cell research.

[13]  H. Mitchison,et al.  PCD Detect: Enhancing ciliary features though image averaging and classification. , 2020, American journal of physiology. Lung cellular and molecular physiology.

[14]  T. Welte,et al.  Generation of two hiPSC clones (MHHi019-A, MHHi019-B) from a primary ciliary dyskinesia patient carrying a homozygous deletion in the NME5 gene (c.415delA (p.Ile139Tyrfs*8)). , 2020, Stem cell research.

[15]  T. Welte,et al.  Generation of two human induced pluripotent stem cell lines (MHHi017-A, MHHi017-B) from a patient with primary ciliary dyskinesia carrying a homozygous mutation (c.7915C > T [p.Arg2639*]) in the DNAH5 gene. , 2020, Stem cell research.

[16]  T. Welte,et al.  Generation of two hiPSC lines (MHHi016-A, MHHi016-B) from a primary ciliary dyskinesia patient carrying a homozygous 5 bp duplication (c.248_252dup (p.Gly85Cysfs*11)) in exon 1 of the CCNO gene. , 2020, Stem cell research.

[17]  B. Scholte,et al.  Generation of an induced pluripotent stem cell line (MHHi018-A) from a patient with Cystic Fibrosis carrying p.Asn1303Lys (N1303K) mutation. , 2020, Stem cell research.

[18]  Hae-Chul Park,et al.  A nonsense variant in NME5 causes human primary ciliary dyskinesia with radial spoke defects , 2020, Clinical genetics.

[19]  Taylor M. Matte,et al.  Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells , 2020, bioRxiv.

[20]  A. Haverich,et al.  Generation of three induced pluripotent stem cell lines (MHHi012-A, MHHi013-A, MHHi014-A) from a family with Loeys-Dietz syndrome carrying a heterozygous p.M253I (c.759G>A) mutation in the TGFBR1 gene. , 2020, Stem cell research.

[21]  J. Favier,et al.  Active mucus–cilia hydrodynamic coupling drives self-organization of human bronchial epithelium , 2019, Nature Physics.

[22]  L. Jan,et al.  Multi-scale spatial heterogeneity enhances particle clearance in airway ciliary arrays , 2019, Nature Physics.

[23]  T. Cantz,et al.  Chemically-Defined, Xeno-Free, Scalable Production of hPSC-Derived Definitive Endoderm Aggregates with Multi-Lineage Differentiation Potential , 2019, Cells.

[24]  Xiaoke Yin,et al.  TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation , 2019, EMBO molecular medicine.

[25]  N. Pedemonte,et al.  High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs , 2019, Stem cell reports.

[26]  A. Horani,et al.  Primary Ciliary Dyskinesia (PCD): A genetic disorder of motile cilia. , 2019, Translational science of rare diseases.

[27]  H. Brunner,et al.  Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects , 2018, American journal of human genetics.

[28]  T. Welte,et al.  Why, when and how to investigate primary ciliary dyskinesia in adult patients with bronchiectasis , 2018, Multidisciplinary Respiratory Medicine.

[29]  L. Yonker,et al.  Expansion of Airway Basal Cells and Generation of Polarized Epithelium. , 2018, Bio-protocol.

[30]  T. Scheper,et al.  Stem Cell Reports Resource Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture , 2018 .

[31]  Michael J. Cronce,et al.  Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function. , 2017, Lab on a chip.

[32]  G. Göhring,et al.  Generation of non-transgenic iPS cells from human cord blood CD34+ cells under animal component-free conditions. , 2017, Stem cell research.

[33]  Fabian Grubert,et al.  Induced Pluripotent Stem Cell Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity , 2017, American journal of respiratory and critical care medicine.

[34]  E. Valente,et al.  Motile and non‐motile cilia in human pathology: from function to phenotypes , 2017, The Journal of pathology.

[35]  Richard Novak,et al.  Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip. , 2016, Cell systems.

[36]  E. Ziętkiewicz,et al.  Aminoglycoside-stimulated readthrough of premature termination codons in selected genes involved in primary ciliary dyskinesia , 2016, RNA biology.

[37]  Robert Zweigerdt,et al.  Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single‐Use Stirred Tank Bioreactors , 2016, Stem cells translational medicine.

[38]  S. Amselem,et al.  RSPH3 Mutations Cause Primary Ciliary Dyskinesia with Central-Complex Defects and a Near Absence of Radial Spokes. , 2015, American journal of human genetics.

[39]  Mandy B. Esch,et al.  TEER Measurement Techniques for In Vitro Barrier Model Systems , 2015, Journal of laboratory automation.

[40]  L. Ostrowski,et al.  Cryo-electron tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia , 2014, Nature Communications.

[41]  Robert Zweigerdt,et al.  Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture , 2014, Stem cell reports.

[42]  H. Omran,et al.  Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia , 2014, European Respiratory Journal.

[43]  S. Ogawa,et al.  Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells , 2014, Stem cell reports.

[44]  J. Shendure,et al.  Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype. , 2014, American journal of respiratory and critical care medicine.

[45]  F. Collins,et al.  Founder Mutation in RSPH4A Identified in Patients of Hispanic Descent with Primary Ciliary Dyskinesia , 2013, Human mutation.

[46]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[47]  E. Ziętkiewicz,et al.  Mutations in Radial Spoke Head Genes and Ultrastructural Cilia Defects in East-European Cohort of Primary Ciliary Dyskinesia Patients , 2012, PloS one.

[48]  Robert Zweigerdt,et al.  Scalable expansion of human pluripotent stem cells in suspension culture , 2011, Nature Protocols.

[49]  Colin A. Johnson,et al.  Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular-pair abnormalities. , 2009, American journal of human genetics.

[50]  E. Gaffney,et al.  Modelling mucociliary clearance , 2008, Respiratory Physiology & Neurobiology.

[51]  S. Antonarakis,et al.  DNAH5 mutations are a common cause of primary ciliary dyskinesia with outer dynein arm defects. , 2006, American journal of respiratory and critical care medicine.

[52]  M. Chilvers,et al.  Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia☆ , 2003, Journal of Allergy and Clinical Immunology.