Universität Ulm Institut für Allgemeine Physiologie Direktor: Prof. Dr. Paul Dietl Recapitulating aspects of alveolar epithelial dysfunction related to idiopathic pulmonary fibrosis utilizing an iPSC-derived air-liquid interface model system Dissertation zur Erlangung des Doktorgrades der Humanbiologie der Medizinischen Fakultät der Universität Ulm Eva Dorothea Schruf Schrobenhausen 2020 Amtierender Dekan: Prof. Dr. Thomas Wirth Erstgutachter: Prof. Dr. Manfred Frick Zweitgutachter: Prof. Dr. Jan Tuckermann Tag der Promotion: 23.07.2020 II TABLE OF CONTENTS List of Abbreviations ........................................................................................................ V List of Figures ................................................................................................................ VIII List of Tables .................................................................................................................... IX 1. Introduction .................................................................................................................... 1 1.1. The alveolar epithelium ............................................................................................. 1 1.1.1. Structure and physiological function of the pulmonary alveolus .................... 1 1.1.2. Human alveolar epithelial cell types in vivo and in vitro ................................. 1 1.1.3. The embryologic origin of alveolar epithelial cells .......................................... 4 1.2. Idiopathic pulmonary fibrosis ..................................................................................... 6 1.2.1. Epidemiology, clinical presentation and pharmacologic management .......... 6 1.2.2. Pathogenesis and risk factors ........................................................................ 7 1.2.3. Pathophysiological remodeling of the lung epithelium in IPF ......................... 9 1.2.4. Current model systems of pulmonary fibrosis .............................................. 11 1.3. Modeling lung development and disease utilizing human pluripotent stem cells .... 12 1.3.1. Embryonic stem cells and induced pluripotent stem cells ............................ 12 1.3.2. Directed differentiation of hPSCs towards lung epithelial cells .................... 14 1.3.3. hPSC-derived models of lung disease ......................................................... 17 1.4. Aims and objectives ................................................................................................ 18 2. Material and Methods .................................................................................................. 19 2.1. Material ................................................................................................................... 19 2.1.1. Human lung samples ................................................................................... 19 2.1.2. Cells ............................................................................................................. 19 2.1.3. Recombinant proteins and small molecules ................................................. 19 2.1.4. Cell culture media and additives .................................................................. 20 2.1.5. Antibodies .................................................................................................... 23 2.1.6. TaqMan Gene Expression Assays ............................................................... 24 2.1.7. Kits, buffers and other reagents ................................................................... 24 2.2. Methods .................................................................................................................. 25 2.2.1. Human cell culture ........................................................................................ 25 2.2.2. LysoTracker Green DND-26 live cell staining .............................................. 27 2.2.3. Cilia beat measurements in primary small airway cultures .......................... 27 2.2.4. Measurement of total cell number and Caspase-3/ 7 activity ...................... 28 2.2.5. Measurement of MMP protein concentrations by ELISA ............................. 28 2.2.6. Flow cytometry ............................................................................................. 28 2.2.7. Immunohistochemistry ................................................................................. 29 2.2.8. Semi-quantitative measurement of SFTPC+ area in iPSC-derived cultures 30 2.2.9. Visual collagen I quantification in primary human lung fibroblast cultures ... 31 2.2.10. Transmission electron microscopy .............................................................. 31 2.2.11. Quantitative Real-Time PCR ....................................................................... 32 III 2.2.12. Molecular karyotyping and PluriTest of iPSC lines ...................................... 32 2.2.13. RNA extraction, Illumina library preparation and RNA-seq .......................... 32 2.2.14. Bioinformatic analysis of RNA-seq data ...................................................... 33 2.2.15. Composition of an IPF-relevant cytokine cocktail (IPF-RC) ......................... 34 2.2.16. Statistical analysis ....................................................................................... 34 3. Results ......................................................................................................................... 35 3.1. Quality control of human iPSC lines ........................................................................ 35 3.2. Optimization of a differentiation protocol to derive ATII-like cells from iPSCs......... 36 3.3. Characterization of iPSC-derived lung epithelial progenitor cells ........................... 43 3.4. Characterization of iPSC-derived ATII-like cells differentiated in ALI culture .......... 47 3.5. Design of a novel IPF-relevant cytokine cocktail to mimic aspects of a pro-fibrotic lung milieu in vitro ................................................................................................... 51 3.6. IPF-RC stimulation of iPSC-derived distal lung epithelial progenitor cells induces an IPF-related phenotype and shifts differentiation towards proximal lineages .......... 54 3.7. IPF-RC treatment of primary small airway basal cells favors MUC5AC+ goblet cell differentiation and impairs ciliation ......................................................................... 62 3.8. The metaplastic epithelium in distal IPF lungs contains atypical transition zones between SFTPC+ ATII-like cells and MUC5B+ goblet-like cells .............................. 63 4. Discussion .................................................................................................................... 65 4.1. Establishment of an iPSC-derived ALI model system of alveolar epithelial differentiation .......................................................................................................... 65 4.1.1. Derivation of NKX2.1+ lung epithelial progenitor cells from human iPSCs ... 65 4.1.2. Maturation of iPSC-derived lung epithelial progenitors towards ATII-like cells…………………………………………………....…………………………..68 4.1.3. Cellular heterogeneity of the established iPSC-derived model system ........ 69 4.1.4. The apparent absence of ATI-like cells in iPSC-derived ALI cultures .......... 70 4.2. Modeling aspects of IPF-related alveolar epithelial dysfunction in vitro utilizing iPSC-derived ALI cultures ...................................................................................... 71 4.2.1. Recapitulating IPF-related processes utilizing iPSC-derived ALI cultures ... 71 4.2.2. The role of individual cytokines in the induction of an IPF-related epithelial phenotype .................................................................................................... 73 4.2.3. Small airway basal cells as a potential source of the bronchiolized epithelium in IPF ............................................................................................................ 74 4.2.4. Limitations concerning the relevance of the established iPSC-derived model system of IPF-related alveolar epithelial dysfunction ................................... 74 4.3. Conclusions and perspectives ................................................................................ 75 5. Summary....................................................................................................................... 78 6. References.................................................................................................................... 79 Appendix .......................................................................................................................... 96 Acknowledgements ....................................................................................................... 100 Curriculum Vitae ............................................................................................................ 101 IV LIST OF ABBREVIATIONS ABCA3 ATP-binding cassette sub-family A member 3 ActA Activin A AFE Anterior foregut endoderm AGER Advanced glycosylation end product–specific receptor ALI Air-liquid interface AQP5 Aquaporin 5 ATI cell Alveolar epithelial type I cell ATII cell Alveolar epithelial type II cell ATRA All-trans retinoic acid BAL Bronchoalveolar lavage BASC Bronchioalveolar stem cell BMP Bone morphogenetic protein BSA Bovine serum albumin Cas9 CRISPR-associated endonuclease Cas9 CAV1 Caveolin 1 CF Cystic fibrosis CFTR Cystic fibrosis transmembrane