Recapitulating Aspects of Alveolar Epithelial Dysfunction Related To

Home , Pulmonary alveolus

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

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

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 conductance regulator CHIR CHIR99021

CO2 Carbon dioxide

CRISPR Clustered regularly interspaced short palindromic repeats

DCI Dexamethasone, 8-Br-cAMP, IBMX DE Definitive endoderm Dor Dorsomorphin ECM Extracellular matrix EMT Epithelial-to-mesenchymal transition

FACS Fluorescent-Activated Cell Sorting FGF10 Fibroblast growth factor 10

FGF2 Fibroblast growth factor 2 (alos known as bFGF)

FMT Fibroblast-to-myofibroblast transformation

FOXJ1 Forkhead box protein J1

FVC Forced vital capacity

H&E Hematoxylin and eosin

HPS Hermansky-Pudlak syndrome hPSC Human pluripotent stem cell

IBMX 3-Isobutyl-1-methylxanthine IL-13 Interleukin 13 IL-1β Interleukin 1 beta IL-33 Interleukin 33 IL-4 Interleukin 4 IL-8 Interleukin 8 ILD Interstitial lung disease IPF Idiopathic pulmonary fibrosis IPF-RC IPF-relevant cocktail iPSC Induced pluripotent stem cells KGF Keratinocyte growth factor (also known as FGF7)

KRT14 Cytokeratin-14

KRT5 Cytokeratin-5

MACS Magnetic-Activated Cell Sorting MACS Magnetic-activated cell sorting

MCP1 Monocyte chemotactic protein 1 (also known as CCL2) MMP-10 Matrix metalloproteinase 10 MMP-7 Matrix metalloproteinase 7 MUC5AC Mucin-5AC

MUC5B Mucin-5B

NKX2.1 NK2 Homeobox 1 (also known as TTF-1) Nog Noggin

O2 Oxygen

PAS Periodic acid-Schiff

PBS Phosphate buffered saline pcw Post-conception week

PDPN Podoplanin (also known as T1α) PF-ILD Progressive fibrosing interstitial lung disease RA All-trans retinoic acid RI ROCK inhibitor (Y-27632) RNA-seq Ribonucleic acid sequencing

SAEC Small airway epithelial cell SB SB431542 SCGB1A1 Secretoglobin family 1A member 1 (also known as Uteroglobin)

SEM Standard error of the mean

SFD Serum-free differentiation SFTPA Surfactant protein A SFTPB Surfactant protein B SFTPC Surfactant protein C SFTPD Surfactant protein D SOX2 SRY-Box Transcription Factor 2 SOX9 SRY-Box Transcription Factor 9 TGF-β1 Transforming growth factor beta 1 TNF-α Tumor necrosis factor alpha TSLP Thymic stromal lymphopoietin VAFE Ventralized anterior foregut endoderm Wnt Wingless-related integration site

VII

LIST OF FIGURES

Figure 1. Microscopic anatomy and cell types of the human alveolus...... 2 Figure 2. Schematic diagram of the stages of human lung development...... 5 Figure 3. Alveolar epithelial remodeling in IPF characterized by dysfunctional re- epithelialization and bronchiolization...... 9 Figure 4. The induced pluripotent stem cell (iPSC) technology...... 13 Figure 5. Pluripotency marker expression in human iPSC lines...... 35 Figure 6. Three germ layer differentiation potential of human iPSC lines...... 36 Figure 7. iPSC differentiation towards definitive endoderm (DE) and anterior foregut endoderm (AFE)...... 37 Figure 8. Influence of BMP antagonism (by Dorsomorphin dihydrochloride or Noggin treatment) during iPSC-derived DE to AFE differentiation on AFE and lung epithelial progenitor cell formation...... 38 Figure 9. Influence of the re-seeding timing and density of iPSC-derived DE cells on lung epithelial progenitor cell differentiation...... 39 Figure 10. Influence of air-exposure on ATII-like cell differentiation from iPSC-derived lung epithelial progenitor cells...... 40 Figure 11. Positive effects of temporal modulation of Wnt signaling on ATII-like cell differentiation from iPSC-derived lung epithelial progenitor cells...... 41 Figure 12. CD47+ MACS sorting of iPSC-derived lung epithelial progenitor cells. iPSC line SFC084-03-01...... 42 Figure 13. Optimized differentiation protocol to derive lung epithelial progenitor cells from iPSCs...... 43 Figure 14. Changes in gene expression during iPSC towards lung epithelial progenitor cell differentiation...... 44 Figure 15. Characterization of lung epithelial progenitor cells derived from iPSC line SFC065-03-03...... 45 Figure 16. Characterization of lung epithelial progenitor cells derived from iPSC line SFC084-03-01...... 46 Figure 17. Differentiation of iPSCs towards ATII-like cells in ALI culture. iPSC line SFC065- 03-03...... 47 Figure 18. Time-course of alveolar epithelial-associated transcript expression during the maturation of iPSC-derived ALI cultures...... 48 Figure 19. Expression of transcripts associated with alveolar and airway epithelial cell fates in day 49 cultures derived from iPSC line SFC065-03-03...... 49 Figure 20. ATII-like phenotypic and functional properties of ALI cultures derived from iPSC line SFC065-03-03...... 50

VIII

Figure 21. Expression of transcripts associated with alveolar and airway epithelial cell fates and ATII-like phenotype of day 49 cultures derived from iPSC line SFC084-03-01...... 51 Figure 22. IPF-RC-induced collagen I formation in primary human lung fibroblasts...... 52 Figure 23. Influence of IPF-RC stimulation on MMP secretion from iPSC-derived lung epithelial cultures...... 54 Figure 24. Effect of IPF-RC treatment on the expression of transcripts known to be deregulated in IPF lungs in iPSC-derived ATII-like cells...... 55 Figure 25. Fibrosis-related transcriptional changes induced by IPF-RC stimulation of iPSC- derived lung epithelial progenitors...... 56 Figure 26. IPF-RC-induced distal-to-proximal shift in gene expression of iPSC-derived lung epithelial cultures...... 57 Figure 27. Epithelial phenotypic changes recapitulating aspects of IPF-related bronchiolization resulting from IPF-RC stimulation during differentiation...... 58 Figure 28. Alterations in the effect of IPF-RC on iPSC-derived ATII-like cell differentiation following removal of single cytokines from the cocktail...... 59 Figure 29. Influence of IL-13 stimulation on iPSC-derived ATII-like cell differentiation. ... 60 Figure 30. Effects of nintedanib treatment on the transcriptional changes induced by IPF- RC stimulation of iPSC-derived ATII-like cells...... 61 Figure 31. IPF-relevant cytokine cocktail (IPF-RC)-induced changes in gene expression in primary human SAEC cultures...... 62 Figure 32. IPF-relevant cytokine cocktail (IPF-RC)-induced phenotypic and functional alterations in SAEC cultures derived from primary human small airway basal cells...... 63 Figure 33. Epithelial transition zones in cystic lesions of IPF patient lungs...... 64

LIST OF TABLES

Table 1. Previous reports on hPSC differentiation into lung epithelial cells...... 14 Table 2. Literature-based composition of an IPF-relevant cytokine cocktail (IPF-RC) ...... 53

Parts of this dissertation have previously been published in The FASEB Journal:

Schruf E, Schroeder V, Le HQ, et al. Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC-derived air-liquid interface model. The FASEB Journal. 2020;34:7825–7846. https://doi.org/10.1096/fj.201902926R

This is an open access article under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs License (CC BY-NC-ND 4.0).

The permission to reuse the full article in this dissertation was obtained from John Wiley and Sons via the Copyright Clearance Center.

X Introduction

1. INTRODUCTION

1.1. The alveolar epithelium

1.1.1. Structure and physiological function of the pulmonary alveolus

Pulmonary alveoli are tiny air sacs that are located at the ends of terminal bronchioles in the lower respiratory tract of the lung. They represent the smallest functional units of respiration and their primary function is the exchange of oxygen and carbon dioxide between the air that is breathed in and the capillary blood. The average human lung contains between 300 million and 600 million alveoli with a mean diameter of 300 µm per alveolus. Alveoli are composed of a single layer of epithelium and are closely associated with an extensive network of capillaries. In the air-blood barrier, a basement membrane layer fuses the alveolar epithelium to the capillary endothelium, linking the respiratory and cardiovascular system. As the close proximity of alveolar air to blood is essential for the rapid diffusion of gases, the thin alveolar walls do not contain muscle and the lung tissue itself cannot actively contract. However, alveoli are surrounded by elastin rich connective tissue that creates elastic recoil upon stretching of the lung tissue. The homeostatic maintenance of the alveolus, particularly of the alveolar epithelium and the alveolar interstitium, is precisely controlled as any change in its microarchitecture affects gas exchange efficiency. (Weibel, 2009)

1.1.2. Human alveolar epithelial cell types in vivo and in vitro

The fundamental function of the alveolar epithelium is to provide an extensive surface for gas exchange. It is composed of alveolar epithelial type I (ATI) and alveolar epithelial type II (ATII) cells (Figure 1). These two cell types were first described by Frank Low who performed electron microscopy of human alveoli more than six decades ago (Low, 1953).

The alveolar surface is created primarily by the thin squamous ATI cells that cover ~95% of the epithelial surface area (Crapo et al., 1983). Their highly flattened shape allows oxygen to diffuse through them and enter the pulmonary microcirculation. In addition, ATI cells likely play important roles in the regulation of alveolar fluid clearance (Johnson et al., 2002). ATI cells are commonly considered terminally differentiated cells, although one recent study reported that ATI cells contribute to the renewal of the ATII cell population following partial pneumonectomy in mice (Jain et al., 2015). Common marker proteins utilized to identify ATI cells include the water channel protein aquaporin 5 (AQP5), the mucin-type glycoprotein podoplanin (PDPN, previously T1α), caveolin 1 (CAV1) as well as advanced glycosylation end product–specific receptor (AGER) (Borok et al., 1998; Newman et al., 1999; Shirasawa et al., 2004; Williams et al., 1996).

ATI cells have proven difficult to isolate and culture in vitro. However, an optimized harvesting approach has been developed that allows for the isolation and in vitro maintenance of rat ATI cells with a high purity (Wang and Hubmayr, 2011). Alternatively, ATI-like cells that have been derived ex vivo from isolated ATII cells have often been substituted for primary ATI cells in in vitro experiments (Dobbs, 1990; Fuchs et al., 2003).

The cuboidal ATII cells, despite covering only a minor part of the alveolar surface area, make up more than half of the total number of alveolar epithelial cells (Crapo et al., 1983). In 1954, Macklin postulated for the first time that ATII cells secrete material that reduces

1 Introduction

Figure 1. Microscopic anatomy and cell types of the human alveolus. (A) Representative hematoxylin and eosin (H&E) staining of an alveolus in a healthy human lung. Scale bar 50µm. (B) Schematic diagram of the human alveolus. Airflow into alveoli through conducting airways lined by small airway epithelial cells (basal cells, club cells, ciliated cells). Alveolar epithelium composed of thin alveolar epithelial type I (ATI) cells covering most of the epithelial surface area and cuboidal alveolar epithelial type II (ATII) cells. Surfactant secretion by ATII cells to lower alveolar surface tension between gas and hydrated epithelial cell surface. Exchange of oxygen (O2) and carbon dioxide (CO2) between the alveolar air and the capillary blood through highly flattened ATI cells and capillary endothelial cells (air-blood barrier) by passive diffusion. Pulmonary interstitial fibroblasts stabilizing alveolar walls by synthesizing extracellular matrix.

surface tension and observed that ATII cells proliferate after lung injury (Macklin, 1954). Subsequent studies confirmed that ATII cells synthesize and secrete pulmonary surface- active material (surfactant), which serves as a protective film for the alveolus (Mason and Williams, 1977). Surfactant is stored in characteristic, lysosome related, secretory organelles termed lamellar bodies and their tightly packed, intraorganellar bilayer membranes are easily recognizable in electron microscopic images (Weaver et al., 2002). Due to their acidic nature, they can also be visualized using fluorescent LysoTracker dyes, which accumulate in lamellar bodies of viable ATII cells (Haller et al., 1998; Van der Velden et al., 2013). Additionally, lamellar bodies contain specific proteins that allow for the identification of ATII cells using immunohistochemistry, e.g. the lipid transporter ABCA3 (Yamano et al., 2001).

2 Introduction

Pulmonary surfactant, which is composed of multiple lipids (~90% by mass) and proteins (~10% by mass), minimizes the surface tension at the alveolar air-liquid interface and thereby facilitates breathing and prevents alveolar collapse. Predominant phospholipid species in mammalian surfactant are phosphatidylcholines (~60-70% by mass), phosphatidylglycerol and phosphatidylinositol. In addition, surfactant includes cholesterol and other neutral lipids. Within the surfactant film of the alveoli, the hydrophilic head domains of the phospholipids face the aqueous phase near the cellular surface, while the hydrophobic tail domains face the luminal air. The minor protein component of surfactant contains the specific surfactant proteins SFTPA, SFTPD, SFTPB and SFTPC. SFTPA and SFTPD are hydrophilic C-type lectin proteins involved in the innate immune response of the alveolar epithelium to foreign pathogens. The hydrophobic proteins SFTPB and SFTPC, on the other hand, are thought to act primarily as structural components and are essential for the biophysical function of surfactant. Importantly, SFTPC is the only surfactant protein that is expressed exclusively in ATII cells of the adult human lung and thereby represents a specific marker for the identification of these cells. The balance between surfactant secretion and removal is achieved mainly by ATII cells that internalize surfactant via endocytosis and recycle its components. However, alveolar macrophages also contribute to this process, e.g. by capturing and degrading inhaled molecules incorporated into surfactant material. (Parra and Perez-Gil, 2015)

In addition to secreting antimicrobial proteins, ATII cells contribute to innate immunity during infection by releasing various chemokines and cytokines, including IL-1β, IL-1α, TNF-α, IL- 6, IL-8 and MCP1, as well as several complement proteins (Mason, 2006; Strunk et al., 1988). Moreover, ATII cells are capable of trans-epithelial sodium transport and thereby regulate the removal of alveolar surface fluid (Mason et al., 1982).

Furthermore, ATII cells act as stem cells in the adult human lung by contributing to alveolar epithelial maintenance and repair (Barkauskas et al., 2013). It is well known that ATII cells proliferate to re-establish a continuous epithelium after damage to ATI cells (Mason and Williams, 1977). However, initially there was ambiguity whether all ATII cells exerted stem cell functions or if only a certain subpopulation of ATII cells was responsible for alveolar epithelial maintenance and repair. Lineage tracing experiments in mice then revealed that certain rare, long-lived, self-renewing ATII cells serve as progenitors for ATI cells in the postnatal lung (Desai et al., 2014). Recently, Zacharias et al. identified an alveolar epithelial progenitor subpopulation within the human ATII lineage that is responsive to wingless- related integration site (Wnt) and fibroblast growth factor (Fgf) signaling and can be isolated based on the expression of the surface marker TM4SF1 (Zacharias et al., 2018). These findings suggest that a specific subpopulation of ATII cells, rather than the ATII cell population as a whole is responsible for alveolar epithelial regeneration in the adult mammalian lung. It has further been demonstrated that activation of Wnt signaling can mediate the recruitment of previously quiescent subpopulations of ATII cells into a progenitor role upon severe lung injury in mice (Nabhan et al., 2018). Alveolar epithelial progenitor cells are therefore viewed as facultative progenitors of the distal lung (Zacharias et al., 2018). It has further been shown that specific fibroblasts residing in close proximity to alveolar epithelial stem cells play a critical role in the maintenance of their stemness, highlighting the importance of the mesenchymal niche in alveolar epithelial self-renewal (Nabhan et al., 2018; Zepp et al., 2017). Based on observations in mice, it has been proposed that bronchioalveolar stem cells (BASCs) co-expressing the alveolar marker SFTPC and the airway club cell marker SCGB1A1, also contribute to alveolar epithelial renewal (Kim et al., 2005). However, another group reported that they could not find any

3 Introduction evidence for a role of BASCs in alveolar epithelial homeostasis or repair (Rawlins et al., 2009). Overall, the relevance of BASCs remains controversial leaving the ATII cell population or subpopulations thereof as the only confirmed source of alveolar epithelial renewal in the adult human lung.

Alveolar epithelial dysfunction is known to play a key role in the pathogenesis of a variety of degenerative, neoplastic as well as neonatal lung diseases (Desai et al., 2014; Whitsett et al., 2015; Winters et al., 2019). Therefore, the establishment of in vitro models that enable the investigation of ATII cell biology in health and disease is highly desirable. In the 1970s, the first protocols for the isolation and in vitro culture of ATII cells from rat or rabbit lung tissue were published (Kikkawa and Yoneda, 1974; Kikkawa et al., 1975). These protocols utilized proteolytic enzymes and density-based sedimentation to separate ATII cells from other lung cell types. Later, similar methodologies were applied to isolate primary human ATII cells (Robinson et al., 1984). Nowadays, fluorescence-activated cell sorting (FACS) of cells expressing pan-epithelial markers (EpCAM, E-Cadherin) or the ATII-specific surface marker HTII-280 is commonly utilized to purify ATII cells from lung cell suspensions (Gonzalez et al., 2010).

A challenge that recurrently arose during early ATII in vitro experiments and remains an issue today is that primary ATII cells rapidly undergo extensive morphological and functional changes in culture (Diglio and Kikkawa, 1977; Dobbs et al., 1985). After a couple of days on tissue culture plastic in medium containing fetal bovine serum, ATII cells lose their cuboidal appearance and become flattened, while the number of lamellar bodies and the expression of surfactant proteins decreases. As the resulting cells also display an induction of ATI-related transcripts (e.g. CAV1), these flattened ATII-derived cells are commonly utilized as a substitute for primary ATI cells in in vitro experiments, as mentioned above (Fuchs et al., 2003). In recent years, this trans-differentiation issue has been partly resolved by growing primary ATII cells as 3D alveolospheres in co-culture with mesenchymal cells allowing for the maintenance of an ATII phenotype for up to 14 days (Barkauskas et al., 2013). One study reported, that prolonged culture periods of up to 21 days could be achieved by optimizing media conditions to preserve the phenotypic characteristics of human ATII cells (Mao et al., 2015). However, it remains challenging to gain access to primary human lung material and to maintain functional ATII cells in culture for sufficient experimental time windows. This particularly complicates the establishment of robust medium- or high-throughput assay systems to investigate the response of human ATII cells to novel pharmacological agents. In addition, the limited proliferative capacity of primary ATII cells in vitro restricts their usability for studies requiring genetic manipulation.

1.1.3. The embryologic origin of alveolar epithelial cells

As outlined in Figure 2, human lung development occurs across five partly overlapping morphological stages termed embryonic, pseudoglandular, canalicular, saccular and alveolar phase (Burri, 1984). All lung epithelial lineages are derived from the endoderm, while the surrounding mesenchyme is derived from the mesoderm layer. During gastrulation, the endoderm forms as one of the three principle embryonic germ layers and derivates into the foregut, midgut and hindgut (Wells and Melton, 1999). Lung development starts with the formation of the two primary lung buds growing from the respiratory diverticulum on the ventral side of the anterior foregut. The primary lung buds form towards the end of post- conception week (pcw) 4 and are characteristic of the embryonic phase of human lung development. The first transcription factor that is known to be activated in the endodermal

4 Introduction lung primordium in rodents is Nkx2.1 (Ikeda et al., 1995; Lazzaro et al., 1991). Nkx2.1 depletion in mice results in severe defects in lung morphogenesis (Minoo et al., 1999). Moreover, NKX2.1 mutations in humans have been associated with respiratory distress syndrome as well as other alterations affecting the thyroid and the brain (Shetty et al., 2014). It has thus been postulated that all cells of the human lung epithelium derive from embryonic NKX2.1+ ventralized anterior foregut endoderm (VAFE) progenitor cells.

Figure 2. Schematic diagram of the stages of human lung development. Embryonic phase (4-7 pcw): Primary lung bud formation on the ventral side of the anterior foregut endoderm. Pseudoglandular phase (5-17 pcw): Branching morphogenesis. Canalicular phase (16-26 pcw): Initiation of alveolar epithelial differentiation and blood capillary formation. Saccular phase (26-36 pcw): Formation of primitive alveoli, emergence of lamellar bodies and expansion of the capillary network. Alveolar phase (36 pcw ~ 3 years): Secondary septation, alveolar epithelial maturation and fusion of the microcapillary network into a single capillary system result in the establishment of functional alveolar units. Pcw = post-conception weeks.

Following the formation of the lung buds, the airway tree develops through growth and dichotomous branching (branching morphogenesis) during the subsequent pseudoglandular phase that spans approximately 5-17 pcw (Isaacson et al., 2017). The distal tips of human pseudoglandular branching structures co-express the transcription factors SOX2 and SOX9. As cells exit the tips they retain SOX2 expression, but stop expressing SOX9 and differentiate towards airway lineages (Nikolic et al., 2017).

The canalicular phase occurs from 16-26 pcw and is characterized by further epithelial branching, increasing size of existing airways and widening of the most distal epithelial regions. It has been proposed that cells leaving the human canalicular stage distal tip initially co-express ATI and ATII markers at low levels, before a switch towards lineage-specific expression occurs as development proceeds (Nikolic et al., 2017). Yet, it has not been conclusively resolved if the human ATI and ATII lineages arise independently, from a common bipotent progenitor, or if ATI cells arise from transdifferentiating ATII cells. During the canalicular phase, a decrease in columnar height of the tip epithelium occurs within the future alveolar airspaces. At approximately 17 pcw, pro-SFTPC first becomes detectable at very low levels and then increases with advancing gestation age (Nikolic et al., 2017). In addition to the initiation of alveolar epithelial differentiation, the surrounding mesenchyme in the distal regions thins and blood capillary formation occurs (deMello and Reid, 2000).

The saccular stage spans 26-36 pcw and is characterized by the formation of terminal saccules (primitive alveoli) clustering at the end of the airways (Nikolic et al., 2018). These thin-walled structures then expand and become more complex by further subdivisions of airspaces. Alveolar epithelial differentiation continues and surfactant-producing lamellar bodies become detectable in differentiating ATII cells (Baritussio et al., 1981; Chander et al., 1986; Van der Velden et al., 2013). This process is accompanied by further expansion of the capillary network.

5 Introduction

Final maturation of the alveolar epithelium and alveolar septation occur during the alveolar phase of lung development. This phase is commonly considered to range from about 36 pcw to approximately 3 years after birth, although it has been suggested that human alveolarization likely continues into later childhood and even adolescence (Narayanan et al., 2012). In the alveolar phase, secondary septation of primitive alveoli and expansion of the microcapillary network, which fuses into a single capillary system, result in the establishment of functional alveolar units (Schittny, 2017).

Overall, a large proportion of our current knowledge about mammalian lung development is based on rodent studies, which have given detailed insights into the molecular regulation of lung epithelial differentiation. Wnt and bone morphogenetic protein (Bmp) signaling, for example, have been identified as essential regulators of the proximo-distal patterning of the murine lung epithelium (Mucenski et al., 2003; Shu et al., 2005; Weaver et al., 1999). Other regulators include the Shh and the Fgf signaling pathways, as well as Notch, which is responsible for the balance between ciliated and secretory cells in the airway epithelium (Morrisey and Hogan, 2010; Volckaert and De Langhe, 2015). In contrast to murine lung development, the exploration of human lung development has been complicated by the lack of access to human fetal tissue. This is particularly the case for the later stages of lung development. However, substantial morphological and molecular differences exist between human and murine lungs, suggesting that certain aspects can only be studied in human cells (Nikolic et al., 2018). For instance, mice and rats are born during the saccular stage, while human birth occurs during the early alveolar stage (Amy et al., 1977; Burri, 1974; Zeltner et al., 1987). Therefore, the mechanisms regulating the late stages of human lung development remain largely elusive.

1.2. Idiopathic pulmonary fibrosis

1.2.1. Epidemiology, clinical presentation and pharmacologic management

Idiopathic pulmonary fibrosis (IPF) is a rare chronic interstitial lung disease (ILD) of unknown cause that affects approximately 5 million people worldwide. The prevalence of IPF is estimated to be slightly greater in men than in women and it occurs primarily in older adults with a mean age at presentation of 66 years (Meltzer and Noble, 2008). IPF patients face a very poor prognosis with a median survival of 3.8 years among adults 65 years of age or older in the US (Raghu et al., 2014).

IPF is characterized by fibrotic remodeling of the lung parenchyma that progresses in a variable and unpredictable manner and results in an irreversible decline in lung function (Ley et al., 2011). The disease initially manifests with symptoms of exertional dyspnea and dry coughing and the diagnosis of IPF presents significant challenges. Many patients don’t experience an appropriate and timely diagnosis due to a delay in seeking medical attention and a high frequency of misdiagnosis (Cosgrove et al., 2018). The course of the disease can be impacted by acute, clinically significant deteriorations in respiratory status of unknown etiology, termed acute exacerbations, with the median survival of IPF patients who experience an acute exacerbation being approximately 3-4 months (Collard et al., 2016).

Diagnosis of IPF requires an exclusion of other causes of ILD, e.g. inhalation of domestic and occupational pollutants, autoimmune disorders or drug toxicity, and the presence of the High-Resolution Computed Tomography pattern of usual interstitial pneumonia (UIP). The

6 Introduction radiologic UIP pattern is characterized by temporal as well as spatial heterogeneity and typically features macroscopic honeycombing, which refers to the presence of clustered cystic airspaces of consistent diameter, with or without peripheral traction bronchiectasis or bronchiolectasis. Histopathologic features of the UIP pattern include a dense fibrosis with architectural distortion that is predominantly distributed subpleurally and/or paraseptally, patchy involvement of lung parenchyma by fibrosis and the presence of fibroblast foci (Raghu et al., 2018).

While non-pharmacologic management strategies, including smoking cessation, supplemental oxygen administration or pulmonary rehabilitation, have long been available to IPF patients to improve their overall quality of life, there was a complete lack of efficacious pharmacologic treatment options until recently (Lederer and Martinez, 2018). In 2014, two drugs, nintedanib (Ovev®) and pirfenidone (Esbriet®), were both shown to slow the rate of forced vital capacity (FVC) decline in IPF patients significantly in placebo-controlled, randomized trials and were then approved for the indication (King et al., 2014; Richeldi et al., 2014). However, despite the approval of these antifibrotic agents, that can slow down disease progression, IPF remains inevitably fatal and lung transplantation is still the only treatment option for IPF patients with respiratory failure that enhances survival (Kumar et al., 2018). This leaves an unmet medical need for a causative pharmacotherapeutic cure that halts or reverses decline in lung function.

1.2.2. Pathogenesis and risk factors

To date, the exact pathophysiological mechanisms underlying IPF remain unknown. However, significant efforts made in recent years that aimed to increase our understanding of these processes, have led to the emergence of conceptual models describing potentially relevant aspects of IPF pathogenesis. A favored model posits that various environmental exposures in combination with age-related and genetic predisposition result in a susceptibility to aberrant wound healing in response to repetitive alveolar epithelial cell micro-injuries (Daccord and Maher, 2016; King et al., 2011; Lederer and Martinez, 2018; Ryu et al., 2014). According to the current consensus in the field that perceives IPF as an epithelial/fibroblastic cross-talk disorder, the abnormally activated alveolar epithelium then secretes mediators that stimulate uncontrolled myofibroblast activation and excessive extracellular matrix (ECM) formation (collagens, hyaluronan, fibronectin) in the lung interstitium (Kang et al., 2016; King et al., 2011; Selman and Pardo, 2002). This excessive ECM deposition is considered a main driver of the initial spreading of fibrotic foci within subpleural areas by changing the mechanical properties of the tissue and resulting in the continuous progression of fibrosis. ECM remodelling thereby plays a pivotal role in IPF pathogenesis not only by causing a progressive stiffening of the lung that leads to a constrain of ventilation, but also by exposing lung cells to mechanical stress (Parker et al., 2014). After years of repetitive injury, during which functional alveoli are gradually replaced with nonfunctional connective tissue, this dysregulated fibro-proliferative repair ultimately results in irreversible scarring, loss of lung function and premature death (Chambers and Mercer, 2015).

While chronic inflammation was also considered a potential hallmark of IPF pathogenesis in the past, the field has gradually shifted away from this paradigm after it became clear that immunosuppressive steroid therapy does not lead to an improvement in clinical outcome in IPF patients, despite effectively reducing inflammation in the lung (Grijm et al., 2005; Raghu et al., 2012). Yet, innate immune cells, such as monocyte-derived alveolar macrophages,

7 Introduction have been shown to be involved in the development of lung fibrosis and the the role of inflammatory dysregulation and the immune system in IPF remains controversial (Bringardner et al., 2008; Misharin et al., 2017).

Interestingly, mechanistic overlaps between IPF and cancer have been described, including similarities in (epi-)genetic changes, altered cell-to-cell communication and a deregulation of specific signalling pathways, including various tyrosine kinase mediated pathways (Vancheri, 2013). Additionally, an aberrant activation of developmental pathways, e.g. Wnt, SHH, Notch and FGF signalling, has been reported in IPF, the progression of which is commonly viewed as a vicious cycle of lung injury and repair (Sakai and Tager, 2013; Selman et al., 2008; Shi et al., 2009).

Importantly, the disease specific milieu within the lung has also been suggested to play a central role in the pathogenesis of IPF (Agostini and Gurrieri, 2006; Xu et al., 2016a). Various potential pro-fibrotic mediators have been identified by analyzing IPF tissue and BAL fluid, including cytokines, growth factors and enzymes involved in ECM remodeling. Among them, transforming growth factor-beta 1 (TGF-β1) is considered a central player in the induction of exaggerated matrix deposition within the IPF lung, mainly through fibroblast recruitment and transformation (King et al., 2011; Selman and Pardo, 2014; Wuyts et al., 2013). Additionally, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and connective tissue growth factor have been described as fibrogenic factors in IPF (Beyer and Distler, 2013; Bonner, 2004; Inoue et al., 2002; Pan et al., 2001). Cytokines upregulated and thought to play prominent roles in IPF include IL-13, IL-1β, IL-8, TNF-α and MCP-1 (Agostini and Gurrieri, 2006). Increased levels of matrix metalloproteinases (MMPs), enzymes that can degrade extracellular matrix components as well as release, cleave and activate various growth factors, cytokines, chemokines and cell surface receptors have also been detected in IPF patients. Growing evidence suggests that some of them, particularly MMP-7, promote a fibrotic response (Pardo et al., 2016).

By definition, a common identifiable cause of lung injury is absent in IPF patients. Yet, while the primary etiology of the disease remains elusive, various potential risk factors have been described. These include older age, cigarette smoking and environmental exposures, such as certain metal or wood dusts among other particles, as well as gastroesophageal reflux and chronic viral infection with Epstein-Barr virus or hepatitis C (Raghu et al., 2011). Interestingly, genetic predisposition has also emerged as a prominent risk factor of IPF in recent years. A single-nucleotide polymorphism (rs35705950) in the MUC5B promoter resulting in overexpression of MUC5B and increased production of the encoded protein mucin-5B in distal airway epithelial cells has been associated with an increased risk of IPF (Nakano et al., 2016; Seibold et al., 2011). Although the potential mechanistic role of this genetic variant remains unknown, it has been hypothesized that alterations in airway mucin composition might lead to aberrant mucociliary clearance and result in changes in the lung microbiome and subsequent immune responses (Evans et al., 2016). Besides this gain-of- function variant of MUC5B, which represents the strongest currently known genetic IPF risk factor, mutations in genes involved in telomere length maintenance, including hTERT and hTR, have been detected in some IPF patients and in familial pulmonary fibrosis (Armanios et al., 2007). Moreover, mutations in surfactant protein genes, including SFTPC and SFTPA2, have been associated with familial ILD, but are rare in sporadic cases of IPF (Raghu et al., 2011). As recent studies suggest that ~70% of IPF cases have a genetic association and as experts in the field have argued that the name IPF no longer accurately represents our current understanding of the disease pathogenesis, it has been proposed

8 Introduction that a change of name should be considered in the future (Dickey and Whitsett, 2017; Wolters et al., 2018).

1.2.3. Pathophysiological remodeling of the lung epithelium in IPF

The myofibroblast has long been recognized as the primary effector cell in IPF by secreting excessive amounts of collagen-rich extracellular matrix in response to pro-fibrotic mediators (Scotton and Chambers, 2007). However, in recent years the alveolar epithelium has attracted increasing attention as a growing body of evidence indicates that the susceptibility of surfactant secreting ATII cells to injury and the resulting defective alveolar repair play a critical role in IPF disease onset and progression (Figure 3).

Figure 3. Alveolar epithelial remodeling in IPF characterized by dysfunctional re-epithelialization and bronchiolization. Representative hematoxylin and eosin (H&E) staining of (A) a healthy lung and (B) an IPF lung. Scale bars 400µm. (C) Schematic diagram showing the healthy alveolar epithelium composed of flat ATI cells and surfactant-producing ATII cells. (D) Schematic diagram summarizing currently known characteristics of the remodeled distal lung epithelium in the course of the obliteration of functional lung alveoli. Ongoing alveolar epithelial injury of unknown cause results in denudation of the basal lamina and fibroblast-to- myofibroblast transformation (FMT); dysfunctional alveolar epithelial cell repopulation and failure to restore epithelial integrity; bronchiolization: impaired proximo-distal patterning characterized by replacement of functional ATI and ATII cells with hyperplastic epithelial cells of an airway-like phenotype; release of pro-fibrotic mediators potentiates the vicious injury-repair-cycle by stimulating proliferation and excessive extracellular matrix (ECM) deposition by myofibroblasts.

ATII cell senescence and apoptosis are characteristic for IPF and other hallmarks of aging, including telomere attrition, epigenetic alterations and mitochondrial dysfunction have been proposed as mechanisms of alveolar epithelial dysfunction and IPF pathogenesis by preventing correct wound-healing in response to repetitive micro-injuries (Barbas-Filho et al., 2001; Selman and Pardo, 2014). A murine study further found that targeted injury and cell death of ATII cells is sufficient to induce fibrotic lung remodeling, supporting the hypothesis of ATII cells as major players in the development of pulmonary fibrosis (Sisson et al., 2010).

As early as 1985, areas of alveolar epithelial denudation as well as defective re- epithelialization of the denuded basal lamina by proliferating pneumocytes were observed in interstitial lung fibrosis and identified as a potential pathogenic mechanism (Katzenstein,

9 Introduction

1985). Since then, various reports have focused on the histopathological characterization of distal epithelial abnormalities in the IPF lung and microscopic honeycombing has emerged as a unique histological feature of UIP. It is distinct from macroscopic radiologic honeycombing and refers to the presence of small cysts that are usually located within densely fibrotic subpleural areas and are often filled with mucus plugs and macrophages (Evans et al., 2016). While some honeycomb cysts, the origin of which remains debatable, are lined entirely by ATII cells, many contain both alveolar epithelial cells and pseudostratified columnar epithelial cells similar to those normally restricted to the conducting airways. This phenotype, characterized by a loss of the normal proximo-distal patterning of the respiratory epithelium and an aberrant emergence of airway epithelial-like cell types within the alveolar regions of the lung, has been termed bronchiolization. A high percentage of mucus cells expressing MUC5B was shown to be present in this bronchiolized distal lung epithelium of IPF patients (Plantier et al., 2011; Seibold et al., 2011). In a subsequent study, Seibold et al. proposed that ATII cells are absent from the pseudostratified airway-like epithelial regions of IPF cystic lesions containing MUC5B+ goblet cells and that these regions are separated from SFTPC+ ATII cells by double- negative (SFTPC-/MUC5B-) epithelial transition zones (Seibold et al., 2013). In addition to mucin producing secretory cells, SCGB1A1+ club cells have been detected within hyperplasic alveolar epithelial regions of diseased lungs (Akram et al., 2013; Buendia- Roldan et al., 2016). Moreover, the expression of cilium-associated genes like FOXJ1 was shown to be linked to microscopic honeycombing in IPF (Yang et al., 2013). Another group found that the amount of KRT5+ airway basal cells was dramatically increased in IPF and identified a novel population of KRT5+/KRT14+ epithelial cells in distal airways and alveolar regions of humans IPF lungs (Smirnova et al., 2016).

The identification of specific bronchoalveolar cells, residing at the duct junction, as putative stem cells of the distal lung in mouse models of lung injury together with the observation of increased numbers of basal cell and club cell populations in IPF lesions have led to the hypothesis that the bronchiolized epithelium in IPF might originate from airway basal cell or BASC migration towards alveolar regions (Akram et al., 2013; Kim et al., 2005; Kumar et al., 2011; Smirnova et al., 2016). However, the role of BASCs in human lung homeostasis and repair remains debatable and many recent reports strongly suggest that a specified Wnt responsive alveolar epithelial stem cell population, constituting a subpopulation of ATII cells residing within a specific mesenchymal niche, serves as the residual stem cell pool of the alveolar epithelium (Barkauskas et al., 2013; Nabhan et al., 2018; Rawlins et al., 2009; Zacharias et al., 2018; Zepp et al., 2017). Interestingly, a recent single-cell RNA-seq study of human IPF lungs highlighted that intermediate cell types reside in bronchiolized regions, co-expressing alveolar and conducting airway cell selective markers (Xu et al., 2016b). In addition to an upregulation of airway-related transcripts such as SOX2, TP63, KRT5, KRT14, MUC5B and SCGB1A1 in ATII cells from IPF patients, the authors reported an induction of genes involved in early lung morphogenesis, including SOX9 and Wnt signaling related transcripts (PRKX, Wnt7b, DKK1, PORCN) in IPF. These findings are indicative of an involvement of aberrant lung epithelial differentiation in IPF-related alveolar epithelial remodeling. Yet, the definite cellular origin of the aberrant epithelium lining the microscopic honeycomb cysts of IPF patients remains unknown.

In addition to the aforementioned processes, a potential involvement of epithelial-to- mesenchymal transition (EMT) has been proposed in IPF. The observations that alveolar epithelial cells that surround fibrotic foci in human IPF lungs co-express epithelial as well as mesenchymal markers, and that murine alveolar epithelial cells can undergo EMT in a

10 Introduction

TGF-β1 overexpression mouse model of lung fibrosis, have led to the hypothesis that alveolar epithelial cells contribute to the myofibroblast population in IPF (Kim et al., 2006; Willis et al., 2005; Yamaguchi et al., 2017). In contrast, another study examined lung epithelial cells and lung fibroblasts from control and IPF donors and found no evidence for a direct contribution of EMT to the myofibroblast population (Gabasa et al., 2017). Thus, the role of EMT in IPF pathogenesis remains controversial.

1.2.4. Current model systems of pulmonary fibrosis

Multiple in vivo and in vitro model systems of fibrotic lung disorders have been developed in an attempt to better understand IPF pathogenesis and to enable the preclinical testing of novel treatment strategies.

In vivo models of lung fibrosis are based on known causes of fibrosis in humans, but none of them fully recapitulates the human UIP pattern of lung fibrosis that is found in IPF patients. Bleomycin-induced pulmonary fibrosis in rodents has been the most commonly used animal model. The anti-neoplastic agent bleomycin which is administered intratracheally, intranasally, intravenously, subcutaneously or intra-peritoneally effects cytotoxicity by inducing DNA breaks and results in reversible fibrosis characterized by severe early inflammation, in contrast to the irreversible, progressive nature of human IPF (Williamson et al., 2015). Other murine in vivo models of pulmonary fibrosis rely on the overexpression of known pro-fibrotic mediators, including TGF-β1, TNF-α or IL-1β, via adenovirus-mediated gene transfer, on irradiation or on FITC, silica or asbestos administration (B Moore et al., 2013).

In addition to the aforementioned rodent models, the use of ex vivo precision-cut lung slices derived from tissue explants has been proposed to facilitate anti-fibrotic drug discovery by bridging the gap between classical in vivo and in vitro models (Lehmann et al., 2018). Due to the rareness of human IPF patient material, stimulation with TGF-β1, TNF-α, PDGF-AB and LPA has been applied to induce fibrotic changes in healthy human lung slices (Alsafadi et al., 2017). Alternatively, healthy murine lungs or bleomycin-treated mouse lungs can serve as the source material the slices are prepared from.

In vitro models of pulmonary fibrosis have historically focused on fibroblast biology and have relied on human fetal lung fibroblast cell lines (e.g. WI-38, MRC-5) or primary adult lung fibroblast cultures. Currently, the TGF-β1-mediated induction of FMT in primary human lung fibroblasts from healthy or IPF donors, with alpha smooth muscle actin and collagen I production as primary readouts, serves as a robust routine assay and has been expanded to a screening platform for anti-fibrotic drug discovery (Weigle et al., 2019). In addition to fibroblast cultures on rigid tissue culture plastic or glass surfaces, mechanically tunable substrates, e.g. collagen I hydrogels of discrete stiffness, have been used to demonstrate how changes in matrix stiffness influence fibroproliferation and contractile function of IPF fibroblasts (Marinković et al., 2013). 3D cultures of fibroblasts in collagen matrices have proven to be a useful tool to study fibroblast contraction during wound healing as they mimic the 3D nature of lung tissue and the resistance of the connective tissue matrix to deformation (Arora et al., 1999). Additionally, decellularized human lung scaffolds have been utilized to demonstrate the influence of fibrotic matrices on myofibroblast differentiation (Booth et al., 2012).

The recognition of epithelial cells as central players in IPF pathogenesis has led to the emergence of a variety of lung epithelial-centric in vitro models. Immortal epithelial tumor

11 Introduction cell lines such as A549, Calu-3 and NCI-H441 have been employed to model processes like TGF-β1-mediated EMT or epithelial barrier breakdown (Togami et al., 2017). In addition to static cultures, advances in bioengineering have led to the development of a novel lung- on-a-chip device that allows for the administration of cyclic stretch to A549 cells, thereby adding a repetitive mechanical stimulus that mimics breathing movements (Felder et al., 2019). While cancer cell lines possess indefinite replication potential and guarantee low variability between experiments, these genomically altered cells fail to closely mimic the physiological functions of the healthy alveolar or airway epithelium. The use of primary bronchial or small airway epithelial cells (SAECs) matured in air-liquid interface (ALI) culture is the current gold standard for studying the airways, as these cultures very closely resemble the in vivo airway epithelium (Pezzulo et al., 2011). The use of primary cells to study the alveolar epithelium, however, has been much more challenging due to the limited access to primary human ATII cells, particularly from diseased patients, their low expandability and the rapid loss of ATII marker expression and function when cultured in vitro (Mao et al., 2015). Therefore, the field is currently facing a lack of suitable human in vitro model systems to investigate alveolar epithelial dysfunction in IPF.

The rising interest in the intercellular crosstalk between fibroblasts, epithelial cells, endothelial cells and immune cells in fibrotic lung diseases has urged the development of more complex model systems, in which multiple cell types and their interactions with each other can be studied simultaneously. In addition to enabling ALI, commercial Transwell systems have proven to be useful for culturing lung epithelial cells with fibroblasts in direct or indirect co-culture with each other. For instance, Epa et al. utilized an indirect Transwell co-culture system to demonstrate that primary small airway epithelial cells are capable of protecting fibroblasts from TGF-β1-mediated FMT (Epa et al., 2015). Prasad, S., et al. showed that primary healthy and IPF lung fibroblasts display distinct migratory responses to scratch-mediated epithelial injury in A549 cells (Prasad et al., 2014). In addition, multicellular 3D organoid systems have been developed to model fibrotic lung disease. One study described the generation of primary IPF pulmospheres, composed of epithelial cells, endothelial cells, macrophages and mesenchymal cells, and found that pulmosphere invasiveness in a 3D matrix in vitro correlated with disease progression in IPF patients (Surolia et al., 2017). Another group reported that human TGF-β1-activated myofibroblasts are less potent in supporting organoid formation of adult mouse lung epithelial cells than untreated fibroblasts (Ng-Blichfeldt et al., 2019).

Within the last few years, the spectrum of available in vitro models to study lung diseases and the role of the alveolar epithelium has expanded due to the emergence of human pluripotent stem cell (hPSC)-derived systems (discussed in detail in 1.3.3.).

1.3. Modeling lung development and disease utilizing human pluripotent stem cells

1.3.1. Embryonic stem cells and induced pluripotent stem cells hPSCs, including embryonic stem cells and induced pluripotent stem cells (iPSCs), can proliferate indefinitely in culture while maintaining the ability to differentiate into any given cell type in the human body.

Thomson et al. isolated the first embryonic stem cell lines from human blastocysts more than 20 years ago (Thomson et al., 1998). In this pioneering study, the authors showed that

12 Introduction these cells had normal karyotypes and maintained the potential to form derivates of all three embryonic germ layers even after months of in vitro culture. The great utility of human embryonic stem cells for developmental biology, drug discovery, and future cell therapy, was recognized early due to their potential to provide a virtually unlimited source of genomically unaltered cells. However, only a limited number of human embryonic stem cell lines have been approved for research and their broad application has been hindered by ethical considerations relating to the use of human embryonic tissue.

In 2006, Takahashi and Yamanaka first demonstrated the induction of iPSCs that were reprogrammed from murine fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4 by retroviral transduction (Takahashi and Yamanaka, 2006). Only one year later, the generation of iPSC lines derived from human somatic cells was described for the first time (Takahashi et al., 2007; Yu et al., 2007). Human iPSC technology provides the great advantage that somatic patient cells can be reprogrammed, followed by differentiation into disease-relevant cell types, making iPSC-derived models extremely powerful tools for in vitro disease modeling of genetic disorders (Soldner and Jaenisch, 2012). While embryonic stem cells, the use of which for research purposes is tightly regulated, have remained the gold standard in pluripotent stem cell research, the emergence of iPSCs has bypassed these ethical issues and has resulted in a dramatic increase in research activities involving pluripotent stem cells.

Figure 4. The induced pluripotent stem cell (iPSC) technology. Reprogramming of somatic cells from healthy or diseased donors into iPSCs via ectopic expression of specific transcription factors (OCT3/4, SOX2, KLF4, cMYC). Differentiation of iPSCs into tissue-specific cell types of all three germ layers (endoderm, mesoderm, ectoderm). Use of iPSC-derived cells for in vitro disease modeling or cell therapy applications.

The development of the hPSC research field was further accelerated by the emergence of novel genome editing tools that allow for efficient gene targeting in hPSCs (Hockemeyer and Jaenisch, 2016). Due to the development of protocols using site-specific nucleases, e.g. CRISPR/Cas9, it is now possible to efficiently knock out or overexpress genes in hPSCs,

13 Introduction as well as to introduce disease relevant mutations or to constitutively express reporter genes. Another major advance in the hPSC field has been the establishment and refinement of directed differentiation regimes in which hPSC differentiation is constrained towards a specific cell type via the step-wise recapitulation of the respective developmental stages and pathways of a certain target tissue in vitro (Murry and Keller, 2008). To date, a multitude of human cell types have been derived from hPSCs, but dramatic differences in differentiation potentials into specific lineages have been detected across different lines (Kim et al., 2011; Osafune et al., 2008). Yet in some cases, hPSC-derived cells can already be employed for drug discovery purposes, particularly to model tissues for which primary cells are not available for in sufficient amounts due to limited accessibility, e.g. cardiomyocytes, neurons, hepatocytes, macrophages, pancreatic cells and podocytes (Benedetti et al., 2018; Ko and Gelb, 2014).

1.3.2. Directed differentiation of hPSCs towards lung epithelial cells

In recent years, there has been significant progress in the lung stem cell field, leading to the establishment of directed differentiation protocols to derive various airway and alveolar epithelial lineages from hPSCs. A summary of previous studies that have reported hPSC differentiation into lung lineages is provided in Table 1.

Table 1. Previous reports on hPSC differentiation into lung epithelial cells.

Cell types generated by Culture format used for differentiation of hPSC-derived airway/ alveolar lineage Reference lung progenitors specification

ATII (SFTPC+) Mouse kidney capsule graft (Green et al., 2011)

basal (TP63+) Mouse subcutaneous graft (Mou et al., 2012)

basal (TP63+, KRT14+) ciliated (FOXJ1+, AcTUB+) 2D ALI culture (Wong et al., 2012) goblet (MUC5AC+) ATII (SFTPC+, SFTPB+) ATI (PDPN+, HOPX+, AQP5+) basal (TP63+) 2D submerged culture (Huang et al., 2014) ciliated (FOXJ1+) goblet (MUC5AC+, MUC5B+) club (SCGB1A1+) ATII (SFTPC+, SFTPB+) ATI (AQP5+) 3D co-culture with fetal (Gotoh et al., 2014) club (SCGB1A1+) human lung fibroblasts basal (KRT5+) basal (TP63+) ciliated (FOXJ1+, AcTUB+, CFTR+) 2D ALI culture (Firth et al., 2014) goblet (MUC5AC+) club (SCGB1A1+)

ATII (SFTPC+, SFTPB+) ATI (PDPN+, HOPX+) basal (TP63+) 3D culture (Dye et al., 2015) ciliated (FOXJ1+, AcTUB) club (SCGB1A1+) Table continued on p. 15

14 Introduction

Continuation of Table 1.

Cell types generated by Culture format used for differentiation of hPSC-derived airway/ alveolar lineage Reference lung progenitors specification basal (KRT5+) ciliated (FOXJ1+, AcTUB+, CFTR+, SNTN+) 3D culture/ ALI culture (Konishi et al., 2016) goblet (MUC5AC+) club (SCGB1A1+) basal (KRT5+, TP63+) 3D culture/ ciliated (FOXJ1+, AcTUB+) transplantation into mouse (Dye et al., 2016) goblet (MUC5AC+) kidney capsule, omentum, club (SCGB1A1+) epididymal fat pad alveolar (SFTPB+, MUC1+, 3D culture ± co-culture with LPCAT1+) and airway (TP63+) fetal mouse lung (Hawkins et al., 2017) lineages mesenchyme ATII (SFTPC+, SFTPB+, ABCA3+, MUC1+) 3D culture/ ATI (CAV1+, PDPN+, HOPX+) (Chen et al., 2017) mouse kidney capsule graft goblet (MUC5AC+, MUC5B+) ciliated (FOXJ1+) basal (KRT5+, TP63+) ciliated (AcTUB+) 3D culture (McCauley et al., 2017) goblet (MUC5AC+) club (SCGB3A2+)

ATII (SFTPC+, SFTPB+) 3D culture (Jacob et al., 2017)

ATII (SFTPC+, SFTPB+, ABCA3+, 3D ± co-culture with fetal SFTPA+, SFTPD+, DCLAMP+) (Yamamoto et al., 2017) human lung fibroblasts ATI (PDPN+) ATII (pro-SFTPC+, SFTPB+, ABCA3+) 3D culture/ ATI (PDPN+, HOPX+) (Miller et al., 2018) mouse airway graft goblet (MUC5AC+, MUC5B+) club (SCGB1A1+) “distal bipotent progenitor” (PDPN+/SFTPC+/SOX9+) 3D culture/ (Chen et al., 2018) ATII (SFTPC+, SFTPB+) mouse kidney capsule graft ATI (PDPN+, AQP5+) ATII (SFTPC+, MUC1+, ABCA3+, SFTPB+, HTII-280+) ATI (PDPN+, CAV1+, HOPX+) basal (KRT5+, TP63+) 3D culture (de Carvalho et al., 2019) ciliated (AcTUB+) goblet (MUC5B+) club (SCGB1A1+) 3D co-culture with fetal ATII (SFTPC+, ABCA3+, CDLAMP+) (Korogi et al., 2019) human lung fibroblasts

While different media compositions and culture methods have been utilized, all published approaches share a common trait in that they aim to recapitulate the different stages of lung development in vitro by applying step-wise signaling cues. In brief, hPSCs are driven

15 Introduction towards a definitive endoderm (DE) fate, followed by anterior foregut endoderm (AFE) formation and differentiation into NKX2.1+ ventralized anterior foregut endoderm (VAFE) cells. In 2011, Green et al. found that a brief period of TGFβ and BMP4 inhibition after definitive endoderm formation was required to induce anterior foregut endoderm specification of hPSCs (Green et al., 2011). This study thereby enabled the derivation of human NKX2.1+ lung epithelial progenitors in vitro. In subsequent reports, NKX2.1+ lung progenitors were either cultured under 2D conditions, under 3D conditions in a matrix with or without additional feeder cells, or transplanted into immunocompromised mice to induce further differentiation towards mature lung epithelial cell types. In addition, ALI culture has been utilized to increase cellular maturation of proximal airway epithelial cells, but as yet has not been applied to alveolar epithelial cells (Firth et al., 2014; Wong et al., 2012). Most studies focused on the generation of airway cell types, until the first robust in vitro protocol to derive alveolar epithelial cells from hPSCs was published in 2014 (Huang et al., 2014). Huang et al. achieved this by culturing lung progenitors in CHIR99021, KGF and FGF10, in combination with dexamethasone, 8-bromo-cAMP and isobutylmethylxanthine (DCI) during the maturation phase, stimulation with which had previously been shown to induce alveolar maturation in fetal mouse lung explants (Gonzales et al., 2002). Recently, it was found that temporal modulation of Wnt signaling promotes distal differentiation of hPSC-derived lung epithelial progenitor cells, by which the yield of alveolar populations can be further increased (McCauley et al., 2017). Co-culture with fetal human lung fibroblasts also seems to have a beneficial impact on the maturation and maintenance of the alveolar lineage (Gotoh et al., 2014; Yamamoto et al., 2017). This corresponds well to the finding that epithelial- mesenchymal crosstalk, mediated via Wnt and FGF signaling, plays a crucial role during lung development (Volckaert and De Langhe, 2015). In addition, many recent reports have shifted their focus towards 3D culture systems, including lung organoids, alveolospheres and bronchospheres. This has led to the successful recapitulation of major aspects of epithelial branching morphogenesis, a key feature of human lung development, in vitro (Chen et al., 2017).

Despite the substantial progress that has been made in the field, hPSC-derived lung models still face certain drawbacks. While our understanding of which growth factors and small molecules can be applied to drive certain cell fate decisions during directed differentiation has improved significantly over the past couple of years, many signaling cues, particularly those coordinating the later phases human lung development, remain elusive. Therefore, the purity and yield of the desired lung epithelial cell types that can be achieved using current protocols still have potential for improvement. Some groups have consequently generated reporter lines to enrich for certain populations, e.g. SFTPC+ ATII cells (Jacob et al., 2017; Yamamoto et al., 2017). In addition, previous reports on hPSC-derived lung cells showed that despite their phenotypic similarities to mature lung epithelium, these models are equivalent to human fetal lung cells, rather than representing an adult state (Chen et al., 2017; Nikolic et al., 2018). This is also observed in other tissue types and currently seen as one of the major limitations of hPSC-derived model systems for toxicological screening or drug testing applications (Baxter et al., 2015; Hrvatin et al., 2014; Koivumaki et al., 2018). The increasing use of lung organoid systems also compromises the utility of hPSC-derived epithelial cultures for these applications, as they don’t allow easy accessibility or physiological air-exposure of the apical surface.

From another perspective, hPSC-derived lung epithelial cultures could constitute highly valuable models to gain insights into the role of stem cells during lung repair and regeneration, not despite but rather due to their immature nature by which they resemble

16 Introduction adult stem cells. Recently, alveolar epithelial progenitors, a Wnt responsive subpopulation of ATII cells, were identified as the alveolar stem cell in the lung (Zacharias et al., 2018). Interestingly, the gene expression profile of AEPs, which regenerate the alveolar epithelium within the adult lung, show enrichment of genes associated with lung development. This is in line with previous reports, highlighting the influence of developmental pathways in lung regeneration (Bertoncello, 2016; Kotton and Morrisey, 2014; Whitsett et al., 2011). Moreover, a role for aberrantly activated developmental pathways has also been described in many lung diseases, including IPF (Sakai and Tager, 2013; Selman et al., 2008; Shi et al., 2009).

Overall, hPSC-derived models hold great promise for the future, as they theoretically grant access to an unlimited amount of disease-relevant cell types, such as ATII cells, for basic research and drug-discovery purposes. These cells could be utilized to establish new robust and reproducible models that are far less prone to donor-based variabilities than primary cell-based models.

1.3.3. hPSC-derived models of lung disease

Human in vitro models are urgently needed to gain a better understanding of lung diseases and to bridge the gap between preclinical animal studies and the clinic in drug development. While the lung epithelium, particularly the epithelium of the alveoli, plays a major role in the pathogenesis of various respiratory diseases, insufficient access and maintainability in culture has prevented the establishment of robust in vitro models using primary ATII cells. Therefore, many hopes have been placed on hPSC-derived lung epithelial cells as a potential cell source for the establishment of novel disease models or regenerative medicine applications. So far, most studies in the field have focused on optimizing the differentiation conditions and yields of hPSC-derived lung epithelial cells. Yet, some first attempts to apply these cells to model disease-relevant processes within the human lung have been made and are introduced in the following paragraphs.

In recent years, there has been a high interest in the generation of iPSC lines from diseased patients for in vitro disease modeling and potential regenerative medicine purposes. Cystic fibrosis (CF) is an autosomal recessive monogenetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In 2012, Mou et al. first produced CF-specific lung progenitor cells from human CF iPSCs (Mou et al., 2012). Two groups then utilized targeted genome editing to correct the CFTR mutation in CF patient- derived iPSCs and demonstrated that epithelial cells derived from the resulting iPSCs expressed mature CFTR protein and displayed restored CFTR chloride channel function (Crane et al., 2015; Firth et al., 2015). The toolbox for iPSC-based CF modelling was expanded further by the establishment of iPSC-derived airway organoids capable of CFTR- dependent forskolin-induced swelling. Functional defects in forskolin-induced swelling in CF-specific iPSC-derived airway organoids could be rescued by a correction of the disease- causing mutation (McCauley et al., 2017).

Similar in vitro gene therapy approaches have been made to correct mutations in the SFTPB gene that result in SFTPB deficiency and congenital lung disease. Jacob et al. showed that CRISPR-based gene correction of iPSCs derived from patients carrying a homozygous SFTPB mutation restored surfactant processing in iPSC-derived ATII cells (Jacob et al., 2017). In another study, Leibel et al. utilized lentiviral infection of iPSCs derived from a patient with SFTPB deficiency to induce exogenous SFTPB expression in lung organoids differentiated from infected cells (Leibel et al., 2019). In addition to genetic diseases, viral

17 Introduction infection with respiratory syncytial virus has been modeled using hPSC-derived lung organoids and was shown to result in swelling and shedding of infected cells (Chen et al., 2017).

Due to the high demand for suitable in vitro models of fibrotic lung diseases, some researchers have focused on the potential application of hPSC-derived cells for the establishment of such systems. One group utilized iPSC-derived mesenchymal organoids and TGF-β1 stimulation to recapitulate fibrosis-related fibroblast activation (Vijayaraj et al., 2019; Wilkinson et al., 2017). Two other laboratories investigated Hermansky-Pudlak syndrome (HPS), a rare genetic disease that is characterized by dysfunction of lysosome- related organelles and manifests itself in interstitial pneumonia among other severe symptoms. Strikoudis et al. focused on mesenchymal alterations in lung organoids derived from hPSCs carrying HPS-associated mutations and observed an upregulation of proteins related to myofibroblast formation compared to wildtype organoids (Strikoudis et al., 2019). Korogi et al., on the other hand, generated alveolar epithelial organoids from patient-specific HPS iPSCs and demonstrated that HPS ATII cells contained abnormally distributed, enlarged lamellar bodies with impaired secretory function compared to gene-corrected isogenic controls (Korogi et al., 2019). Apart from the aforementioned studies focusing on rare monogenetic forms of pulmonary fibrosis, no hPSC-derived model systems that can robustly recapitulate aspects of fibrotic lung disease or that are suitable for investigating the role of epithelial cells in this context, have been established.

1.4. Aims and objectives

The unmet need for human in vitro systems to investigate alveolar epithelial dysfunction currently represents a major obstacle for the development of mechanism-specific therapeutics for various respiratory indications. Therefore, the establishment of novel cell culture models for this purpose would be of high interest, particularly to study diseases like IPF, in which a critical involvement of ATII cells has been highlighted. Recently, directed differentiation of iPSCs has emerged as a novel tool that could potentially serve as an unlimited source for any given cell type of the human body. The main aim of this thesis was to apply this technology to establish a novel human iPSC-derived in vitro model of alveolar epithelial dysfunction in IPF.

Thus, the first objective of this work was to establish a protocol to derive ATII-like cells from iPSCs. In an initial set of experiments, optimization of a directed differentiation protocol was conducted to generate NKX2.1+ lung epithelial progenitor cells from iPSCs, followed by maturation into SFTPC+ ATII-like cells in ALI culture, mimicking the physiological environment of the lung epithelium. Subsequently, RNA-seq, qRT-PCR, flow cytometry, immunohistochemistry and transmission electron microscopy analyses were conducted to evaluate the phenotype of the derived cultures.

The second goal of this study consisted of the application of this platform to the development of a novel IPF-relevant disease model. As a first step, lung epithelial progenitor cells were generated utilizing the previously established directed differentiation approach. iPSC- derived ALI cultures were then treated with a pro-fibrotic cytokine cocktail to mimic aspects of an IPF-relevant lung milieu in vitro. Subsequently, in depth characterization of the stimulated cultures by RNA-seq, qRT-PCR, ELISA and immunohistochemistry was carried out to verify the ability of this model system to recapitulate IPF-related processes in vitro and to assess the potential effect of a pro-fibrotic environment on alveolar epithelial differentiation.

18 Material and Methods

2. MATERIAL AND METHODS

2.1. Material

2.1.1. Human lung samples

Formalin-fixed and paraffin embedded lung samples were purchased from Folio Biosciences (Powell, OH, US) under the regulatory conditions of the Boehringer Ingelheim corporate policy regarding the acquisition and use of human biospecimen. IPF patient samples were reviewed internally by a trained pathologist (on H&E basis) and the initial diagnosis of IPF was confirmed.

2.1.2. Cells

The human iPSC lines utilized in this study were obtained from the StemBANCC consortium and were reprogrammed from human skin fibroblasts via non-integrating Sendai virus: o SFC065-03-03 EBiSC: STBCi057-A; Biosamples ID: SAMEA104493762 o SFC084-03-01 EBiSC: STBCi033-A; Biosamples ID: SAMEA104493681 o SFC086-03-01 EBiSC: STBCi052-A; Biosamples ID: SAMEA104493741

Primary human cells were obtained from Lonza (Basel, Switzerland): o Primary human small airway epithelial cells CC-2547; Lot 501937 o Primary human lung fibroblasts CC-2512; Lot. 0000608197

2.1.3. Recombinant proteins and small molecules

Activin A Tocris 338-AC-050 All-trans retinoic acid (ATRA) Sigma Aldrich R2625-100MG BMP4 Tocris 314-BP cAMP (8-Br-cAMP) Sigma Aldrich B5386-25MG CHIR99021 Axon Medchem Axon1386 Dexamethasone Sigma Aldrich D2915 Dorsomorphin dihydrochloride Tocris 3093 FGF10 R&D 345-FG-025 FGF2 Tocris 233-FB-025 IBMX (3-Isobutyl-1-methylxanthine) Sigma Aldrich I5879-100MG IL-13 R&D Systems 213-ILB-005 IL-1β R&D Systems 201-LB-005 IL-33 R&D Systems 3625-IL-010 IL-4 R&D Systems 204-IL-010 IL-8 R&D Systems 208-IL-010

19 Material and Methods

IWP-2 Tocris 3533/10 KGF (=FGF7) R&D Systems 251-KG-010 MCP1 (CCL2) R&D Systems 279-MC-010 Noggin Tocris 6057-NG-100 SB431542 Tocris 1614/1 TGF-β1 R&D Systems 240-B-002 TNF-α R&D Systems 210-TA-020 TSLP R&D Systems 1398-TS-010 Wnt3A Tocris 5036-WN Y-27632 Abcam Ab120-129

2.1.4. Cell culture media and additives

0,05% Trypsin/ 0,53 mM EDTA Thermo Fisher MT-25-051-CI 0,5M EDTA pH8 Life Technologies 15575020 1-thioglycerol (MTG) Sigma-Aldrich M6145-25ML B-27 Supplement (50X), serum free Gibco 17504044 Bovine Albumin Fraction V Solution (7.5%) Life Technologies 15260037 Collagen I, Rat Tail Corning 354236 DMEM/F12, GlutaMAX Gibco 31331028 Fetal Bovine Serum, heat inactivated (HI Gibco 10082147 FBS) FGM-2 SingleQuot kit suppl. & growth Lonza CC-4126 factors Fibroblast basal medium Lonza CC-3131 Ham's F-12 Nutrient Mix, GlutaMAX Gibco 31765092 Heparin Solution Stemcell Technologies 7980 Hydrocortisone Stock Solution Stemcell Technologies 7926 IMDM, GlutaMAX Gibco 31980030 L-Ascorbic acid Sigma A4403-100MG Matrigel hESC-qualified Corning 354277 mFreSR Stemcell Technologies 5854 mTeSR1 medium Stemcell Technologies 5850 N-2 Supplement (100X) Gibco 17502001 Pen/Strep Life Technologies 15140122 PneumaCult™-ALI2 Medium Kit (Basal Medium, 10x supplements V2, maintenance Stemcell Technologies 05099 supplements)

20 Material and Methods

PneumaCult™-Ex Plus Medium Kit (Basal Stemcell Technologies 05040 Medium, 50x supplements) StemPro Accutase Gibco A1110501

Media recipes for optimized iPSC to ATII-like cell differentiation protocol

SFD basal medium 375 mL IMDM, Glutamax

125 mL Hams F12 + Glutamax

3.75 mL Bovine Albumin Fraction V Solution (7.5%)

5 mL B-27 Supplement

2.5 mL N-2 Supplement

5 mL Pen/Strep

SFD+ medium (add to SFD basal medium)

50 µg/ mL L-ascorbic acid

0,04 µl/ mL 1-thioglycerol (MTG)

Differentiation media (add to SFD+ medium)

Day 0 10 µM Y-27632

10 ng/ mL Wnt3a

3 ng/ mL BMP4

Day 1 - 3 10 µM Y-27632

100 ng/ mL Activin A

0,5 ng/ mL BMP4

5 ng/ mL FGF2

Day 4 100 ng/ mL Noggin

10 µM SB431542

10 µM Y-27632

Day 5 1 µM IWP2

10 µM SB431542

Day 6 - 12 3 μM CHIR99021

10ng/ mL KGF (=FGF7)

21 Material and Methods

10ng/ mL FGF10

10ng/ mL BMP4

50 nM Retinoic Acid

Day 13-15 3 μM CHIR99021

10ng/ mL KGF (=FGF7)

10ng/ mL FGF10

10ng/ mL BMP4

50 nM Retinoic Acid

10 µM Y-27632

Day 16- 25 3 μM CHIR99021

10ng/ mL KGF (=FGF7)

10ng/ mL FGF10

Day 26 - 34 3 μM CHIR99021

10ng/ mL KGF (=FGF7)

10ng/ mL FGF10

25 ng/ mL Dexamethasone

0,1 mM IBMX

0,1 mM cAMP

Day 35 - 41 10ng/ mL KGF (=FGF7)

10ng/ mL FGF10

25 ng/ mL Dexamethasone

0,1 mM IBMX

0,1 mM cAMP

Day 42 - 49 3 μM CHIR99021

10ng/ mL KGF (=FGF7)

10ng/ mL FGF10

25 ng/ mL Dexamethasone

0,1 mM IBMX

0,1 mM cAMP

22 Material and Methods

2.1.5. Antibodies

Primary antibodies:

ABCA3 (rabbit polyclonal) abcam ab99856 Collagen type I (mouse IgG1) Sigma-Aldrich SAB4200678 E-Cadherin (rabbit IgG) Cell Signaling 3195S FOXA2 (goat IgG) R&D systems AF2400 KRT5 (guinea pig polyclonal) Progen GP-CK5 MUC5AC (mouse IgG1k) Invitrogen MA1-38223 MUC5B (rabbit polyclonal) Sigma-Aldrich HPA008246 NKX2.1/ TTF1 (mouse IgG1k) Invitrogen MA5-13961 NKX2.1/ TTF1 (rabbit IgG) Seven Hills WRAB-1231 Pro-SFTPC (rabbit polyclonal) Millipore AB3786 SFTPB (rabbit polyclonal) Seven Hills WRAB-48604 SFTPC (rabbit IgG) Sigma-Aldrich HPA010928 SOX9 (rabbit IgG) Sigma-Aldrich HPA001758

Conjugated primary antibodies:

KRT5 (rabbit IgG) Alexa Fluor 647 conjugated abcam ab193895 NKX2.1/ TTF1 (rabbit IgG) Alexa Fluor 488 abcam ab196470 conjugated CD47 (human IgG1) FITC conjugated Miltenyi Biotec 130-101-344

Secondary antibodies:

Donkey anti goat IgG Alexa Fluor 488 conjugated Invitrogen A-11055 Donkey anti rabbit IgG Alexa Fluor 594 conjugated Invitrogen A-21207 Donkey anti rat IgG Alexa Fluor 488 conjugated Invitrogen A-21208 Goat anti guinea pig IgG Alexa Fluor 568 conjugated Invitrogen A-11075 Goat anti mouse IgG Alexa Fluor 488 conjugated Invitrogen A-11029 Goat anti mouse IgG1 Alexa Fluor 568 conjugated Invitrogen A-21124 Goat anti mouse IgG Alexa Fluor 594 conjugated Invitrogen A-11032 Goat anti rabbit IgG Alexa Fluor 488 conjugated Invitrogen A-11034 Goat anti rabbit IgG Alexa Fluor 568 conjugated Invitrogen A-11011 Goat anti rabbit IgG Alexa Fluor 647 conjugated Invitrogen A-21245 Goat anti rat IgG Alexa Fluor 594 conjugated Invitrogen A-11007

23 Material and Methods

2.1.6. TaqMan Gene Expression Assays

ABCA3 Applied Biosystems Hs00184543_m1 ACTA2 Applied Biosystems Hs00426835_g1 AGER/ RAGE Applied Biosystems Hs00542584_g1 AQP5 Applied Biosystems Hs00387048_m1 BPIFB1 Applied Biosystems Hs00264197_m1 CAV1 Applied Biosystems Hs00971716_m1 CDH1 Applied Biosystems Hs01023895_m1 COL1A1 Applied Biosystems Hs00164004_m1 FOXA2 Applied Biosystems Hs00232764_m1 FOXJ1 Applied Biosystems Hs00230964_m1 GAPDH Applied Biosystems Hs02758991_g1 ID2 Applied Biosystems Hs04187239_m1 KRT5 Applied Biosystems Hs00361185_m1 MMP10 Applied Biosystems Hs00233987_m1 MMP7 Applied Biosystems Hs01042796_m1 MUC5AC Applied Biosystems Hs01365616_m1 MUC5B Applied Biosystems Hs00861595_m1 NKX2.1/ TTF1 Applied Biosystems Hs00968940_m1 PDPN Applied Biosystems Hs00366766_m1 POU5F1 Applied Biosystems Hs00999632_g1 SCGB1A1 Applied Biosystems Hs00171092_m1 SFTPB Applied Biosystems Hs00167036_m1 SFTPC Applied Biosystems Hs00161628_m1 SOX17 Applied Biosystems Hs00751752_s1 SOX2 Applied Biosystems Hs01053049_s1 SOX9 Applied Biosystems Hs00165814_m1 TM4SF1 Applied Biosystems Hs01547334_m1 TP63 Applied Biosystems Hs00978340_m1

2.1.7. Kits, buffers and other reagents

4% Paraformaldehyde Solution in PBS Boster Bio AR1068 Antibody dilution solution DCS AL120R500 Bovine serum albumin (BSA) Sigma-Aldrich A2153-50G Caspase-Glo 3/7 Assay Kit Promega G8090

24 Material and Methods

Cellfix BD Biosciences 340181 Cytofix fixation buffer BD Biosciences 554655 DEPC-treated water Ambion AM9920 Dimethyl sulfoxide Sigma Aldrich D2650 Donkey serum Sigma-Aldrich D9663 Dulbecco's phosphate-buffered saline Gibco 14040-091 Goat serum Sigma-Aldrich G9023 High-Capacity cDNA Reverse Transcription Applied Biosystems 4368814 Kit Human Total MMP-10 DuoSet ELISA R&D Systems DY910 Human Total MMP-7 DuoSet ELISA R&D Systems DY907 LysoTracker Green DND-26 Life Technologies L7526 MagMAX™-96 Total RNA Isolation Kit Life Technologies AM1830 ProLong Diamond Antifade Mountant with Life Technologies P36962 DAPI QuantiFast Probe PCR +ROX Vial Kit Qiagen 204356 RNeasy Plus Mini Kit Qiagen 74136 Sodium Citrate H.I.E.R. BioLegend 928502 Sterile water Sigma Aldrich W3513-100ml autoMACS Running Buffer Miltenyi Biotec 130-091-221 anti-FITC MicroBeads Miltenyi Biotec 130-048-701 MACS LS columns Miltenyi Biotec 130-042-401 QuadroMACS Separator Miltenyi Biotec 130-090-976

2.2. Methods

2.2.1. Human cell culture

2.2.1.1. Human induced pluripotent stem cell (iPSC) culture iPSC lines were cultured in mTeSR 1 (Stemcell Technologies, Vancouver, Canada) on hESC-qualified Matrigel (Corning, New York, US) coated cell culture plates at 37 °C/ 5%

CO2 in a humidified normoxic incubator and passaged using 0.5 mM EDTA pH 8.0.

2.2.1.2. Differentiation of human iPSCs into lung epithelial progenitor cells

Directed differentiation of iPSCs towards NKX2.1+ lung progenitor cells was performed in serum-free differentiation (SFD+) medium, as previously described (Huang et al., 2015; Huang et al., 2014). Briefly, definitive endoderm (DE) formation of embryoid bodies was induced in the presence of Activin A, followed by dissociation and replating and anterior foregut endoderm (AFE) induction by BMP, TGF-β and Wnt inhibition. Ventralization was achieved by applying Wnt, BMP, FGF and RA signaling. After 14 days of differentiation, ventralized anterior foregut endoderm (VAFE) cells were detached as clumps and either

25 Material and Methods frozen down for later usage or replated and expanded for 10 additional days on hESC- qualified Matrigel (Corning, New York, US) coated cell culture plates.

2.2.1.3. Branching organoid formation of iPSC-derived lung epithelial progenitor cells

Branching of expanded VAFE cells was induced in 3D culture, similar to previous reports (Chen et al., 2017). Cells were cultured in SFD+ medium supplemented with 3 µM CHIR99021 (Axon Medchem, Groningen, Netherlands), 10 ng/ mL rhKGF (R&D Systems, Minneapolis, MN, US) and 10 ng/ mL rhFGF10 (R&D Systems, Minneapolis, MN, US). On day 24 of differentiation, lung progenitor cells were briefly digested with warm 0.05% Trypsin/ 0.53 mM EDTA and detached as clumps, which were collected at the bottom of a canonical tube via sedimentation and plated onto low attachment dishes. The following day, the resulting organoids were embedded into cold growth factor reduced Matrigel (Corning, New York, US) on 48-well tissue culture plates. Following solidification of the Matrigel, the organoids were covered with medium and cultured for up to 35 days. The medium was changed every two days.

2.2.1.4. iPSC-derived lung epithelial progenitor maturation into ATII-like cells in 2D air- liquid interface culture

On day 24 of differentiation, lung progenitor cells were briefly digested with warm 0.05% Trypsin/ 0.53 mM EDTA and detached as clumps and collected via sedimentation. The cells were plated onto hESC-qualified Matrigel (Corning, New York, US) coated Transwell permeable support inserts (PET membrane, 12 well format, pore size 0.4 µm, Corning, New York, US) and cultured under submerged conditions until day 32 to allow the cells to attach and to spread out over the whole insert surface. Subsequently, the apical medium was removed and the cells were cultured at air-liquid interface until day 49 of differentiation. Similar to a previous study, temporal withdrawal of Wnt signaling was employed to promote distal lung epithelial progenitor differentiation towards ATII-like cells (Jacob et al., 2017). From day 24 to day 35 of differentiation, lung progenitors were cultured in SFD+ medium supplemented with 3 µM CHIR99021 (Axon Medchem, Groningen, Netherlands), 10 ng/ mL rhKGF (R&D Systems, Minneapolis, MN, US), 10 ng/ mL rhFGF10 (R&D Systems, Minneapolis, MN, US), 25 ng/ mL dexamethasone (Sigma-Aldrich, St. Louis, MO, US), 0.1 mM 8-Br-cAMP (Sigma-Aldrich, St. Louis, MO, US) and 0.1 mM 3-Isobutyl-1- methylxanthine (Sigma-Aldrich, St. Louis, MO, US). Subsequently, the cells were treated with the same medium, but without CHIR99021, from day 35 to 42, before 3 µM CHIR99021 was added back for one more week until day 49 of differentiation. The culture medium was exchanged every other day during the whole maturation phase. Where indicated in the text, additional treatments were applied during differentiation from day 35 to day 49.

2.2.1.5. ATI-like differentiation of iPSC-derived ATII-like cells

ATI-like differentiation of iPSC-derived ATII cells was induced by replating cells on plastic in 2D submerged culture, as previously described (Jacob et al., 2017). On day 49 of differentiation ATII-like cells were dissociated using Gibco StemPro Accutase cell dissociation reagent (Life Technologies, Carlsbad, CA, US) and replated onto 6-well tissue culture plates in Gibco DMEM Glutamax (Life Technologies, Carlsbad, CA, US) supplemented with 10% FBS (Life Technologies, Carlsbad, CA, US). Media was changed every other day and cells were harvested for analysis after five days. Controls were not

26 Material and Methods replated and remained at air-liquid interface on Transwell permeable support inserts until they were harvested on day 54 of differentiation.

2.2.1.6. Primary human lung fibroblast culture

Primary human lung fibroblasts were grown in fibroblast basal medium (FBM) (Lonza, Basel, Switzerland) supplemented with FGM-2 SingleQuot Kit Supplements & Growth Factors

(Lonza, Basel, Switzerland) at 37 °C and 5% CO2. Cells were passaged maximum 10 times before use.

2.2.1.7. Primary human small airway epithelial cell culture

Primary human Small Airway Epithelial Cells (SAECs) were thawed into a T-175 tissue culture flask and expanded for four days in PneumaCult-Ex Plus Medium (Stemcell Technologies, Vancouver, Canada). A single cell suspension was then created using an Animal Component-Free Cell Dissociation Kit (Stemcell Technologies, Vancouver, Canada) and 90000 cells/ cm2 were seeded onto rat tail collagen type I (Corning, New York, US) coated Transwell permeable support inserts (PET membrane, 24 well format, pore size 0.4 µm, Corning, New York, US). Four days post seeding, the apical medium was removed and the cells were differentiated at air-liquid interface (ALI) in PneumaCult-ALI 2 medium (Stemcell Technologies, Vancouver, Canada) for 28 days. The culture medium was exchanged every other day. Where indicated in the text, IPF-RC treatment was applied to SAEC cultures from 1 to 28 days post-ALI.

2.2.2. LysoTracker Green DND-26 live cell staining iPSC-derived ATII-like cells were dissociated using Gibco StemPro Accutase cell dissociation reagent (Life Technologies, Carlsbad, CA, US) and replated onto hESC- qualified Matrigel (Corning, New York, US) coated Nunc 8-well Lab-Tek Chambered Coverglasses (Thermo Fisher Scientific, Waltham, MA, US) in SFD+ medium, supplemented with 3 µM CHIR99021 (Axon Medchem, Groningen, Netherlands), 10 ng/ mL rhKGF (R&D Systems, Minneapolis, MN, US), 10 ng/ mL rhFGF10 (R&D Systems, Minneapolis, MN, US), 25 ng/ mL dexamethasone (Sigma-Aldrich, St. Louis, MO, US), 0.1 mM 8-Br-cAMP (Sigma-Aldrich, St. Louis, MO, US), 0.1 mM 3-Isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO, US) and 10 µM Y-27632 (Abcam, Cambridge, UK). The cells were allowed to attach overnight, before 100 nM LysoTracker Green DND-26 (Invitrogen, Life Technologies, Carlsbad, CA, US) was added into the medium and the cells were incubated for 20 min at 37 °C protected from light. The medium was then removed and the samples were washed once with prewarmed Gibco FluoroBrite DMEM (Life Technologies, Carlsbad, CA, US), followed by a 5 min incubation in 5 ng/ mL Hoechst 33342 (Life Technologies, Carlsbad, CA, US) in FluoroBrite. Subsequent to three additional washes with FluoroBrite DMEM, fresh FluoroBrite DMEM was added to the cells and the samples were immediately imaged using a Plan-Apochromat 63x oil-immersion objective on an LSM 710 confocal microscope system (Carl Zeiss, Oberkochen, Germany).

2.2.3. Cilia beat measurements in primary small airway cultures

Cilia beat frequency was calculated from image stacks of 2D+time. Six evenly distributed regions of a Transwell insert were imaged using the 32x objective of an Axiovert 25

27 Material and Methods microscope (Zeiss, Oberkochen, Germany) and an acA1300-200µm black and white USB- 3.0 high speed camera (Basler, Ahrensburg, Germany). Ciliary movement was recorded at 100 frames per second for a total of 6 seconds per region. Applications for image capture and analysis were developed using the Halcon 13.0.2 machine vision software toolbox (MVTec Software, Munich, Germany). Visualization of image stack was performed with Analyze (AnalyzeDirect, Overland Park, KS, US). A grey value time course was calculated for each pixel over 512 frames per region. The area covered by motile cilia was determined by quantifying the percentage of pixels with a measurable beating frequency within a region. The mean ciliary beating frequency of a region was determined by calculating the average frequency of the change in grey values.

2.2.4. Measurement of total cell number and Caspase-3/ 7 activity

The total cell number per insert of iPSC-derived day 49 cultures was assessed by dissociating the cultures with Gibco StemPro Accutase cell dissociation reagent (Life Technologies, Carlsbad, CA, US) and counting of cells with a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, US). Caspase-3/ 7 activity of day 49 cultures was measured using a Caspase-Glo 3/7 Assay Kit (Promega, Madison, WI, US) according to the manufacturer’s instructions.

2.2.5. Measurement of MMP protein concentrations by ELISA

MMP-7 and MMP-10 protein concentrations in cell culture supernatants were determined using a Human Total MMP-7 DuoSet ELISA (R&D Systems, Minneapolis, MN, US) and a Human Total MMP-10 DuoSet ELISA (R&D Systems, Minneapolis, MN, US) according to the manufacturer’s instructions.

2.2.6. Flow cytometry

Lung epithelial progenitors were dissociated using Gibco StemPro Accutase cell dissociation reagent (Life Technologies, Carlsbad, CA, US) and taken up into an excess of ice cold 1% BSA in PBS. All subsequent washing steps were performed by applying ice cold wash buffer (0.1% BSA in PBS) and pelleting at 300g for 5min. 1*10^6 cells were transferred into a FACS tube and washed, before prewarmed Cytofix fixation buffer (BD Biosciences, Franklin Lakes, NJ, US) was added and the sample was incubated for 10 min at 37 °C. Cells were then washed and permeabilized in 80% MeOH for 5 min, followed by 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MO, US) in wash buffer for 10 min. Blocking was performed in 10% goat serum (Sigma-Aldrich, St. Louis, MO, US) in PBS for 10 min. Cells were washed and incubated in the respective antibody solution for 30 min in the dark. Unstained controls were incubated in wash buffer, isotype controls were incubated in 1 µg/ mL Alexa Fluor 488 conjugated rabbit IgG Isotype Control (Abcam, Cambridge, UK) and samples for NKX2.1 staining were incubated in 1 µg/ mL Alexa Fluor conjugated anti-TTF1 antibody (Abcam, Cambridge UK). The cells were then washed, resupended in Cellfix (BD Biosciences) and transferred into fresh FACS tubes through a 35 µm strainer cap. Samples were measured on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, US) and analyzed using the FACSDiva (BD Biosciences, Franklin Lakes, NJ, US) and the FloJo v10.6.1 software (FlowJo LLC, Ashland, OR, US).

28 Material and Methods

2.2.7. Magnetic-activated cell sorting

Magnetic-activated cell sorting (MACS) of CD47+ cells was performed on day 24 of differentiation. Lung epithelial progenitors were dissociated using Gibco StemPro Accutase cell dissociation reagent (Life Technologies, Carlsbad, CA, US). Magneting labeling was carried out according to the manufacturer’s instructions using an anti CD47-FITC antibody at a dilution of 1:11 in ice cold autoMACS Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by an incubation in anti-FITC MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were separated using MACS LS columns and a QuadroMACS Separator (Miltenyi Biotec, Bergisch Gladbach, Germany). The column was removed from the mangetic separator and CD47+ labeled cells were flushed out by applying the plunger. The cells were then plated onto matrigel coated Transwell inserts in Day 16- 25 media supplemented with 10 µM Y-27632.

2.2.8. Immunohistochemistry

2.2.8.1. Hematoxylin and eosin and Alcian Blue/ PAS stainings

Transwell cultures were fixed for 30 minutes with 4% Paraformaldehyde (BosterBio, Pleasanton, CA, US) at room temperature and the whole membrane was cut out of the insert for further processing. Fixed inserts were dehydrated and embedded into paraffin following standard procedures. Transwell insert cross-sections or sections of paraffin- embedded IPF lung samples of 3 µm thickness were prepared and rehydrated using a descending series of ethanol. Hematoxylin and eosin (H&E) and Alcian Blue/ Periodic acid- Schiff (PAS) stainings were performed according to standard protocols. Images were acquired with an AxioCam MR3 (Zeiss, Oberkochen, Germany) using the 20x objective of an Axio Imager Z1 (Zeiss, Oberkochen, Germany).

2.2.8.2. Immunofluorescence of iPSC-derived and primary cells

For immunofluorescence of lung epithelial progenitor cells, VAFE cell clumps were replated and expanded on hESC-qualified Matrigel (Corning, New York, US) coated glass bottom plates (12-well, No. 1.5 coverslip, 14 mm glass diameter, MatTek, Ashland, MA, US). On day 24 of differentiation, cells were fixed for 15 min with 4% Paraformaldehyde (BosterBio, Pleasanton, CA, US) at room temperature. The fixed samples were washed with PBS and blocked for one hour at room temperature in 5% BSA with 0.3% TritonX-100 (Sigma-Aldrich, St. Louis, MO, US) in PBS. The cells were then incubated with the respective primary antibodies diluted in antibody dilution solution (DCS, Hamburg, Germany) over night at 4 °C. On the following day, the samples were washed with PBS and incubated with the respective fluorophore conjugated secondary antibodies, diluted 1:500 in antibody dilution solution, for one hour at room temperature. To visualize the nuclei, the cells were then stained with 2 µg/ mL Hoechst 33342 (Life Technologies, Carlsbad, CA, US) in PBS for 5 min and washed with PBS. Subsequently, the finished samples were then covered with 1 mL PBS and imaged.

For immunofluorescence of cells on Transwell inserts, iPSC-derived ATII-like cells or primary SAEC cultures were fixed for 30 minutes with 4% Paraformaldehyde (BosterBio, Pleasanton, CA, US) at room temperature. The inserts were then washed with PBS and the whole membrane was cut out of the insert for further processing.

29 Material and Methods

Whole insert stainings were performed after blocking the fixed cells on the membrane for one hour at room temperature in 5% BSA with 0.3% TritonX-100 (Sigma-Aldrich, St. Louis, MO, US) in PBS, by incubating with primary antibodies diluted in antibody dilution solution (DCS, Hamburg, Germany) over night at 4 °C. After washing, the samples were incubated with the respective fluorophore conjugated secondary antibodies, diluted 1:500 in antibody dilution solution for one hour at room temperature. The membranes were then washed in PBS, mounted with Invitrogen ProLong Diamond Antifade Mountant with DAPI (Life Technologies, Carlsbad, CA, US), coverslipped and imaged.

For staining of insert cross-sections and definitive endoderm bodies, fixed specimen were dehydrated using a Tissue Tek VIP processor and embedded into paraffin blocks. Sections of 3 µm thickness were prepared, deparaffinized with xylene and rehydrated using a descending series of ethanol. Antigen retrieval was performed for 30 min at 95 °C in H.I.E.R. sodium citrate solution (BioLegend, San Diego, CA, US). The samples were blocked for one hour at room temperature with 5% normal goat or normal donkey serum (Sigma-Aldrich, St. Louis, MO, US) in PBS and incubated over night at 4 °C with the respective primary antibodies diluted in antibody dilution solution (DCS, Hamburg, Germany). The samples were washed in PBS with 0.5% Tween-20 (Sigma-Aldrich, St. Louis, MO, US) and incubated with the respective fluorophore conjugated secondary antibodies, diluted 1:500 in antibody dilution solution, for one hour at room temperature. Following washing of the slides with PBS with 0.5% Tween-20 (Sigma-Aldrich, St. Louis, MO, US), the sections were mounted with Invitrogen ProLong Diamond Antifade Mountant with DAPI (Life Technologies, Carlsbad, CA, US) and coverslipped.

All specimens were imaged with a LSM 710 confocal microscope system (Zeiss, Oberkochen, Germany) with an AXIO Observer Z1 using a Plan-Apochromat 20x objective or a Plan-Apochromat 40x water-immersion objective.

2.2.8.3. Immunohistochemistry of IPF lung sections

Immunohistochemistry in IPF patient samples was carried out on the automated Leica IHC Bond-RX platform (Leica Biosystems, Nussloch, Germany) using the Opal method (Perkin Elmer, Waltham, MA, US). Three µm thick sections of FFPE IPF lung tissue on super frost plus slides were deparaffinised and rehydrated for multiplex immunohistochemistry staining. Antigen retrieval was performed for all primary antibodies by heating the sections in Bond ER solution 1 buffer (Leica Biosystems, Nussloch, Germany) at 95°C; pH 6.0 for 20 min. Primary antibodies were diluted with Leica Primary Antibody Diluent (Leica Biosystems, Nussloch, Germany) and sections were incubated with them for 30 min at room temperature followed by incubation (10 min at RT) with Opal Polymer Anti-Rabbit HRP Kit (ARR1001KT; Perkin Elmer Waltham, MA, US). Immunofluorescent signal was visualized using the OPAL (Perkin Elmer, Waltham, MA, US) TSA dye 570 and TSA dye 650 and counterstained with Spectral DAPI (Akoya, Menlo Park, CA). Microscopy of IPF lung samples was conducted with an AxioImager M2 microscope (Zeiss, Oberkochen, Germany) and images were created using an AxioScan scanner and ZEN 2.3 slidescan software (Carl Zeiss, Oberkochen, Germany).

2.2.9. Semi-quantitative measurement of SFTPC+ area in iPSC-derived cultures

Immunofluorescence of fixed whole Transwell inserts was performed as described above. All samples of an experiment were stained and imaged on the same day. Nine randomly

30 Material and Methods selected areas per insert (425.1 µm x 425.1 µm per area) were captured using a 20x Plan- Apochromat objective on a LSM 710 confocal microscope system (Zeiss, Oberkochen, Germany) with the following settings: Alexa Fluor 488 channel, 5% laser power, master gain 600, acquisition speed 9, resolution 1024 px x 1024 px. To account for unevenness in specimen surface and thickness, Z-stack imaging of 20 vertical stacks per area was applied and maximum intensity projection was performed using the ZEN 2012 Black Edition software (Zeiss, Oberkochen, Germany). Image analysis was then performed using the Fiji (Fiji Is Just ImageJ) software (Schindelin et al., 2012). Each image was first converted to 8- bit format and then converted to binary at a threshold of 20-255. In the following, particles within areas >80 px were analyzed and summarized per image. For each analyzed Transwell insert, the mean positive area per image was calculated from the nine selected areas.

2.2.10. Visual collagen I quantification in primary human lung fibroblast cultures

Primary human lung fibroblasts were plated in a poly-D-lysine coated 384 CellCarrier microtiter plate from PerkinElmer in fibroblast basal medium (FBM) with FGM-2TM Single Quots (Lonza, Basel, Switzerland) at a density of 1000 cells per well. After 24 h, the medium was replaced by the same medium containing no serum (starvation medium). 48 h after cell seeding, the starvation medium was replaced with starvation medium containing a mixture of Ficoll 70 and 400 (GE Healthcare, Chicago, IL, US; 37.5 mg/ mL and 25 mg/ mL, respectively), 200 µM vitamin C and IPF-RC (1:1000 dilution). After 72 h, the cell culture medium was removed and cells were fixed with 100% ice-cold methanol for 30 minutes. Next, cells were washed with PBS, permeabilized for 20 minutes using 1% Triton-X-100 (Sigma-Aldrich, St. Louis, MO, US), washed and blocked for 30 minutes with 3% BSA in PBS. After an additional wash step, cell nuclei were stained with 1 µM Hoechst 33342 (Life Technologies, Carlsbad, CA, US) and collagen I was stained using a monoclonal antibody (SAB4200678, Sigma-Aldrich, St. Louis, MO, US). For primary antibody detection, cells were washed and incubated for 30 minutes at 37 °C with Goat anti mouse IgG1 Alexa Fluor 568 secondary antibody. After secondary antibody removal, cells were stained with Invitrogen HCS Cell Mask Green stain (1:50000, Life Technologies, Carlsbad, CA, US). Following a final wash step, images were acquired in an InCell 2200 Analyzer (GE Heathcare, Chicago, IL, US), using 2D-Deconvolution for nuclei (Hoechst channel), cells (FITC channel), and collagen I (TexasRed channel), and images were transferred to the Columbus Image Storage and Analysis system (Perkin Elmer, Waltham, MA, US).

Image analysis was performed as previously described (Schruf et al., 2019; Weigle et al., 2019). Briefly, using the building blocks of the Columbus 2.8.2 Image Data Storage & Analysis system (PerkinElmer, Waltham, MA, US), first nuclei acquired with the Hoechst channel were detected using the building block (BB) “nuclei”. Second, cells were defined with the BB “find cytoplasm” from the FITC channel. Collagen I area was defined by two individual BBs “find simple image region” based on images acquired in the TexasRed channel. Collagen I readouts were normalized to the number of cells per image field. Total number of cells, and total collagen area/total number of cells were used as parameters to quantify effects of IPF-RC.

2.2.11. Transmission electron microscopy

For electron microscopy of iPSC-derived ATII-like cells, Transwell inserts were pre-fixed for 1 h with 4% Paraformaldehyde. The PET membrane with pre-fixed cells was then washed

31 Material and Methods with a 0,1% cacodylate buffer solution for 30 minutes. Afterwards, the samples were transferred into a EM-TP Tissue processor (Leica Microsystems, Wetzlar, Germany) for automated post-fixation, staining, dehydration and embedding. In detail, the samples underwent the following steps: 20 min in 0,1% cacodylate buffer, 3 h in 1% Daltons osmiumtetroxide aq., 3 times 15 min in 0,1% cacodylate buffer solution, 15 min in 30% isopropanol, 30 min each in 30%, 50%, 70%, 90% and 100% isopropanol, and three times 1 h in 100% isopropanol. Following dehydration, sample infiltration with Epoxy resin was achieved as follows: 30 min in 50% isopropanol/ 50% EPON, 30 min in 33% isopropanol/ 66% EPON, 30 min in 20% isopropanol/ 80% EPON and 60 min in 100% EPON. The samples were then incubated twice for 6 h in 100% EPON and hardened at 60°C for 24 hours. Ultra-thin sections (50 nm) were prepared on an Ultracut UCT ultra-microtome (Leica Microsystems, Wetzlar, Germany) and imaged on a TEM 912AB (Zeiss, Oberkochen,Germany).

2.2.12. Quantitative Real-Time PCR

Cells were lysed in RLT Plus buffer (Qiagen) and RNA isolation was performed using the RNeasy Plus Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. In some experiments, human lung total RNA (AM7968, Lot.1850512, Invitrogen, Life Technologies, Carlsbad, CA, US) was used as a positive control. Reverse transcription was performed using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, US) according to the manufacturer’s instructions. 2µl (5 ng) of cDNA were added to a final reaction volume of 10 µl containing the QuantiFast Probe PCR +ROX Vial Kit (Qiagen, Venlo, Netherlands) master mix and the respective Applied Biosystems TaqMan Gene Expression Assay FAM (Life Technologies, Carlsbad, CA, US). qPCR was performed in 384-format using an Applied Biosystems ABI ViiA 7 real- time PCR System (Life Technologies, Carlsbad, CA, US). Gene expression was normalized to GAPDH control and fold change in gene expression relative to iPSCs (day 0 of differentiation) was calculated using the 2^(-ΔΔCT) method. A table of all TaqMan Gene Expression Assays used in this work is provided in the Material section.

2.2.13. Molecular karyotyping and PluriTest of iPSC lines

Molecular karyotyping and bioinformatics assays for pluripotency testing of iPSC lines were performed by external service partners. Molecular karyotyping of iPSC lines SFC065-03- 03, SFC084-03-01 and SFC086-03-01 was performed by LIFE & BRAIN GmbH (Bonn, Germany). PluriTests of iPSC lines SFC065-03-03, and SFC084-03-01 were performed by Thermo Fisher Scientific (Waltham, MA, US).

2.2.14. RNA extraction, Illumina library preparation and RNA-seq

Total RNA of iPSC-derived day 0, day 24 and day 49 cultures (three biological replicates per timepoint) was extracted using the Ambion Magmax-96 total RNA isolation kit (Life Technologies, Carlsbad, CA, US) according to the manufacturer’s instructions. Nucleic acids were captured onto magnetic beads, treated with DNase and total RNA was eluted in 50 μl elution buffer. RNA quality and concentration were measured using an RNA Pico chip on a Bioanalyzer (Agilent, Santa Clara, CA, US).

A sequencing library was prepared using the TrueSeq RNA Sample Prep Kit v2-Set B (Illumina, San Diego, CA, US) with 200 ng of total RNA input per sample, resulting in an

32 Material and Methods average fragment size of 275 bp including adapters. Before sequencing, eight individual libraries were normalized and pooled together using the adapter indices supplied by the manufacturer. Pooled libraries were then clustered on an Illumina cBot Instrument using the TruSeq SR Cluster Kit v3—cBot—HS (Illumina, San Diego, CA, US). Sequencing was performed as 50 bp, single reads and 7 bases index read on an Illumina HiSeq2000 instrument using the TruSeq SBS Kit HS- v3 (50-cycle) (Illumina, San Diego, CA, US).

2.2.15. Bioinformatic analysis of RNA-seq data

2.2.15.1. Step-by-step RNA-seq pipeline

A step-by-step bioinformatics pipeline was utilized to process and technically validate the RNA-seq data. The pipeline was described in detail in a previous study (Sollner et al., 2017). Sequenced read quality was checked with FastQC (v0.11.2). RNA-Seq reads from all samples were aligned to the genome using the STAR Aligner (v2.5.2a11) (Dobin et al., 2013). Duplication rates of the RNA-Seq samples were computed with bamUtil (v1.0.11) to mark duplicate reads and the dupRadar v1.4 Bioconductor R package for assessment (Sayols et al., 2016). The gene expression profiles were quantified using Cufflinks software version 2.2.1 to get the Reads Per Kilobase of transcript per Million mapped reads (RPKM) as well as read counts from the featureCounts software package (Liao et al., 2014; Trapnell et al., 2013).

2.2.15.2. Detection of differentially expressed genes

Comparative analysis was done using the limma R-package (Ritchie et al., 2015). Benjamini-Hochberg correction was used to adjust for multiple testing.

Heatmaps

A heatmap of the top 500 genes deregulated in iPSC-derived day 49 cultures versus day 24 cultures was created utilizing the heatmap3 package of R statistical software and the following cut-off values: q-val < 0.001, |logFC| > 2, maxRPKM > 10.

2.2.15.3. Principle component analysis (PCA)

PCA was performed as previously described, using ClustVis (Metsalu and Vilo, 2015). The top 5000 transcripts according to the coefficient of variation were selected for analysis.

2.2.15.4. Gene set enrichment analysis (GSEA)

Normalized expression data for day 49 +IPF-RC and day 49 samples were analyzed using the GSEA method (Subramanian et al., 2005). Genes were ranked by magnitude of correlation with a class distinction, and the GSEA algorithm determined whether members of a gene set tended to occur towards the top or bottom of the list, and calculated an enrichment score. In order to remove extremely low or not at all expressed transcripts from the data set, NGS data was filtered according to an RPKM value > 2 in at least one of the samples. Gene sets that were significantly associated with the day 49 +IPF-RC class were identified from the C2.CP.REACTOME (Curated) collection of gene sets in the Molecular Signatures Database v6.2 (MSigDB), which comprises 674 gene sets (Liberzon et al., 2015). The null distributions used to calculate the statistical significance of the enrichment scores

33 Material and Methods were generated by permutation of gene sets for the day 49 +IPF-RC vs. day 49 contrast (3 replicates/class). Adjustment for multiple hypothesis testing was performed by determination of FDR. Enrichment scores with an FDR q-value <0.05 were considered to be significant.

2.2.15.5. Calculation of intersections with IPF lung RNA-seq dataset

Expression data for day 49 +IPF-RC and day 49 samples were compared to publicly available data for human IPF and healthy lung tissue (Kusko et al., 2016). Transcripts used for calculating the intersections were selected according to being expressed (at least one sample showing an RPKM >2) in both data sets. This resulted in a universe of 12365 transcripts. De-regulated transcripts were defined by exhibiting a q-value <0.05 and an absolute log ratio of >0.5. Resulting gene sets were split into up- and down-regulated and the intersection of the corresponding gene sets from both data sets was calculated. Significance of the intersection was tested via a hypergeometric test.

2.2.16. Composition of an IPF-relevant cytokine cocktail (IPF-RC)

An IPF-relevant cytokine cocktail (IPF-RC) was designed based on literature studies that compared both IPF and healthy control bronchoalveolar lavage or sputum samples (Table 1). Nine cytokines were selected, based on being significantly upregulated in IPF samples compared to healthy controls. To approximately account for dilution in saline during sample collection, cytokine concentrations one order of magnitude higher than measured in IPF patient bronchoalveolar lavage (BAL) were estimated as suitable for in vitro experiments. Long-term stimulation of cells with IPF-RC was performed to simulate the cytokine milieu present in an IPF lung. Final human recombinant cytokine concentrations (all acquired from R&D Systems, Minneapolis, MN, US) in the cell culture medium during IPF-RC treatment were 300 pg/ mL TGF-β1, 10 pg/ mL IL-1β, 100 pg/ mL TNF-α, 1500 pg/ mL IL-8, 700 pg/ mL MCP1, 40 pg/ mL IL-33, 100 pg/ mL TSLP, 2500 pg/ mL IL-13 and 160 pg/ mL IL-4, respectively. IPF-RC was prepared as a 1000x stock in 0.1% BSA in PBS.

2.2.17. Statistical analysis

All qRT-PCR, ELISA, FACS and image analysis data are presented as mean with error bars representing the SEM of at least three independent experiments (n=3), unless indicated otherwise. Experiments comparing two conditions only were analyzed using two-tailed unpaired Student’s t-test, when variances were homogeneously distributed, or Mann- Whitney U-test, when data were not normally distributed. Experiments containing three or more conditions were assessed using two-way ANOVA followed by Tukey post-hoc test. Data analysis was performed using GraphPad Prism 8.0 (Graph Pad Software Inc., San Diego, CA, US). Significance values are marked as * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

34 Results

3. RESULTS

3.1. Quality control of human iPSC lines

Initially, quality control of human iPSC lines was performed to confirm genome integrity and pluripotency. Molecular karyotyping of lines SFC065-03-03, SFC084-03-01 and SFC086- 03-01 was carried out at LIFE & BRAIN GmbH and no larger chromosomal aberrations were detected. In addition, a PluriTest of iPSC lines SFC065-03-03 and SFC084-03-01 was carried out at Thermo Fisher Scientific and revealed a clear pluripotency expression signature for both lines. These results obtained from analyses by external service providers are shown in the Appendix of this thesis.

Figure 5. Pluripotency marker expression in human iPSC lines. Immunofluorescence of iPSC cultures stained against OCT4, SOX2, NANOG, SSEA4, TRA-1-60 or TRA-1-81. Nuclei stained with DAPI. (A) iPSC line SFC065-03-03. (B) iPSC line SFC084-03-01. (C) iPSC line SFC086-03-01. Scale bars 50µm. Representative immunofluorescence images are shown.

35 Results

Expression of pluripotency markers was also assessed on protein level by immunofluorescence. SFC065-03-03, SFC084-03-01 and SFC086-03-01 cells stained positively for OCT4, SOX2, NANOG, SSEA4, TRA-1-60 or TRA-1-81 (Figure 5). Moreover, iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01 were capable of differentiating into SOX17+ endodermal, HAND1+ mesodermal and OTX+ ectodermal cells, confirming their three germ layer differentiation potential (Figure 6).

Figure 6. Three germ layer differentiation potential of human iPSC lines. Immunofluorescence of iPSCs differentiated into each of the three germ layers utilizing the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, SC027). Endodermal cells stained against SOX17. Mesodermal cells stained against HAND1. Ectodermal cells stained against OTX2. Nuclei stained with DAPI. (A) iPSC line SFC065-03- 03. (B) iPSC line SFC084-03-01. (C) iPSC line SFC086-03-01. Scale bars 50µm. Representative images are shown.

3.2. Optimization of a differentiation protocol to derive ATII-like cells from iPSCs

To derive human ATII-like cells from iPSCs, a directed differentiation protocol was established as described below. As experiments in this sub-chapter were conducted for the purpose of protocol optimization, some conditions were not reproduced in multiple independent differentiation rounds and therefore constitute preliminary data. Following optimization, an in-depth characterization of the main differentiation stages was performed utilizing the finalized protocol (see 3.3. and 3.4.).

Step-wise differentiation of iPSCs towards NKX2.1+ lung progenitor cells was performed as previously described and each differentiation step was optimized for the three iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01 (Huang et al., 2015; Huang et al., 2014). Initially, Activin A was utilized to induce definitive endoderm (DE) formation in 3D culture (Figure 7A). After 4.5 days, cells derived from all three iPSC lines were positive for SOX17, indicative of DE differentiation (Figure 7B). DE cells were dissociated and seeded in 2D adherent culture, before inhibition of BMP, TGF-β and Wnt signaling was applied via

36 Results

Dorsomorphin, SB431542 and IWP-2 stimulation (Figure 7A). This resulted in the formation of FOXA2+ anterior foregut endoderm (AFE) cells in all three iPSC lines (Figure 7C).

Figure 7. iPSC differentiation towards definitive endoderm (DE) and anterior foregut endoderm (AFE). iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01. (A) Differentiation of iPSCs towards AFE via DE formation. RI = Y-27632; ActA = Activin A; SB = SB431542; Dor = Dorsomorphin. (B) Representative immunofluorescence images of DE cells (Day 4.5; 3D culture) stained against SOX17. Nuclei stained with DAPI. Scale bars 100µm. (C) Representative immunofluorescence images of AFE cells (Day 7) stained against FOXA2. Nuclei stained with DAPI. Scale bars 200µm.

AFE cell differentiation into VAFE cells, which constitute early lung progenitor cells during development, was induced by applying Wnt, BMP, FGF and RA signaling. VAFE cells were then expanded for ten additional days in the presence of CHIR99021, FGF10 and KGF.

As it is known that during AFE formation individual iPSC lines respond differently to the application of distinct BMP inhibitors and that this can have implications on later differentiation stages, Dorsomorphin dihydrochloride (Dor) and Noggin (Nog) stimulation were directly compared during AFE differentiation (Figure 8A). qRT-PCR analysis showed that both stimulants resulted in a downregulation the DE-specific transcripts SOX17 and CXCR4 in AFE cultures compared to DE cultures in all three iPSC lines (Figure 8B). Likewise, expression of the pluripotency marker OCT4 was reduced compared to iPSC cultures for both conditions. However, only Nog-treated cultures displayed an upregulation of SOX2 expression in day 7 cultures compared to DE cultures in all three iPSC lines,

37 Results indicative of AFE differentiation. Moreover, preliminary immunofluorescence data from day 25 cultures indicated a higher induction efficiency of NKX2.1+ lung epithelial progenitor cells in Nog-treated cultures compared to cultures stimulated with Dor, in all three iPSC lines (Figure 8C). Therefore, Nog rather than Dor was utilized to antagonize BMP signaling during AFE formation in all consecutive experiments.

Figure 8. Influence of BMP antagonism (by Dorsomorphin dihydrochloride or Noggin treatment) during iPSC-derived DE to AFE differentiation on AFE and lung epithelial progenitor cell formation. iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01. Dor = Dorsomorphin dihydrochloride; Nog = Noggin. (A) Step-wise differentiation of iPSCs towards lung epithelial progenitor cells and comparison of cultures stimulated with Dor or Nog during AFE formation (green). RI = Y-27632; ActA = Activin A; SB = SB431542; RA = all-trans retinoic acid; CHIR = CHIR99021. (B) Gene expression of pluripotency, DE and AFE markers in iPSC (day 0), DE (day 4.5) and AFE cultures stimulated with Dor (day 7 Dor) or Nog (day 7 Nog) normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. (C) Representative immunofluorescence images of day 25 cultures stimulated with Dor (day 25 Dor) or Nog (day 25 Nog) during AFE differentiation stained against lung epithelial progenitor marker NKX2.1. Nuclei stained with DAPI. Scale bars 200µm.

38 Results

The timing and density of DE re-seeding for AFE formation, parameters that have previously been shown to have a major impact on lung epithelial differentiation efficiency, were optimized for iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01. To determine the optimal conditions for each iPSC line, AFE formation was initiated after 4 or 4.5 days respectively and DE cells were re-seeded at a low seeding density of 0.66*10^5 cells/cm2 or a high seeding density of 1,32*10^5 cells/ cm2 (Figure 9A, B). The effect on the lung epithelial progenitor cell induction efficiency was determined by flow cytometry analysis of NKX2.1+ cells in day 24 or day 25 cultures, respectively (Figure 9C). DE induction for 4 days, followed by re-seeding at a high density resulted in the highest percentage of NKX2.1+ cells for all three iPSC lines (55.2% for SFC065-03-03, 54.9% for SFC084-03-01 and 50.9% for SFC086-03-01). Therefore, this condition was chosen for all further experiments.

Figure 9. Influence of the re-seeding timing and density of iPSC-derived DE cells on lung epithelial progenitor cell differentiation. iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01. (A) Step-wise differentiation of iPSCs towards lung epithelial progenitor cells and comparison of 4 days versus 4.5 days DE differentiation (green) and re-seeding of DE cells for AFE formation at different cell densities. RI = Y-27632; ActA = Activin A; SB = SB431542; Dor = Dorsomorphin; Nog = Noggin; RA = all-trans retinoic acid; CHIR = CHIR99021. (B) Morphology of 3D DE cultures after 4 or 4.5 days of differentiation shown by light microscopy. Scale bars 1000µm. (C) Flow cytometry analysis of NKX2.1+ cells on day 24/ day 25. Percentage of NKX2.1+ cells is indicated. Low seeding density = 0.66*10^5 DE cells/cm2. High seeding density = 1,32*10^5 DE cells/ cm2. Data from n=1 differentiation round are shown for each cell line.

Maturation of iPSC-derived lung epithelial progenitor cells towards alveolar epithelial cells was initially carried out in terminal differentiation medium containing CHIR99021, FGF10, KGF, dexamethasone, 8-Br-cAMP and IBMX under submerged conditions as previously

39 Results described (Huang et al., 2015; Huang et al., 2014). In order to mimic a more physiological environment, lung epithelial progenitor cells were seeded onto Transwell inserts and exposed to air during the last two weeks of alveolar epithelial cell maturation (Figure 10A). In all three iPSC lines, air-liquid interface (ALI) culture resulted in higher expression levels of ATII-specific SFTPC by day 49 of differentiation compared to submerged controls (Figure 10B). These results are consistent with a positive influence of air exposure on ATII cell differentiation. Therefore, maturation at ALI was carried out in all further differentiation rounds.

Figure 10. Influence of air-exposure on ATII-like cell differentiation from iPSC-derived lung epithelial progenitor cells. iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01. (A) Differentiation of iPSCs towards ATII-like cells and comparison of submerged and air-liquid interface culture conditions from day 32 of differentiation onwards. RI = Y-27632; ActA = Activin A; SB = SB431542; Dor = Dorsomorphin; Nog = Noggin; RA = all-trans retinoic acid; CHIR = CHIR99021. DCI = dexamethasone, 8-Br-cAMP, IBMX. Sub. = submerged culture on regular cell culture plates. TW sub. = submerged culture on Transwell inserts. TW ALI = air-liquid interface culture on Transwell inserts. (B) SFTPC expression in sub., TW sub. and TW ALI cultures on day 49 of differentiation normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=1 differentiation round. (C) SFTPC expression in TW ALI cultures from day 0 to day 49 of differentiation normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=1 differentiation round. (D) Representative immunofluorescence images of day 49 TW ALI cultures stained against pro-SFTPC. Nuclei stained with DAPI. Scale bars 50µm (left) and 10µm (right).

40 Results

Relative SFTPC expression increased over time during ATII-like differentiation of iPSC- derived lung epithelial progenitor cells for the iPSC lines SFC065-03-03 and SFC084-03- 01. In contrast to this, line SFC086-03-01 only displayed a very moderate induction of SFTPC expression that did not increase further after day 32 of differentiation (Figure 10C). Moreover, immunofluorescence of day 49 ALI cultures revealed that pro-SFTPC+ cells were present in cultures derived from iPSC lines SFC065-03-03 and SFC084-03-01, but not in cultures derived from line SFC086-03-01 (Figure 10D). All consecutive experiments were carried out in iPSC line SFC065-03-03 or SFC084-03-01 due to their proven potential to differentiate towards SFTPC expressing ATII-like cells under the described culture conditions.

Figure 11. Positive effects of temporal modulation of Wnt signaling on ATII-like cell differentiation from iPSC-derived lung epithelial progenitor cells. iPSC line SFC084-03-01. (A) Step-wise differentiation of iPSCs towards ATII-like cells with (+CHIR) or without (-CHIR) the addition of CHIR99021 to the culture medium from day 35 to day 42 of differentiation. RI = Y-27632; ActA = Activin A; SB = SB431542; Dor = Dorsomorphin; Nog = Noggin; RA = all-trans retinoic acid; CHIR = CHIR99021. DCI = dexamethasone, 8-Br-cAMP, IBMX. (B) Gene expression of SFTPC, SFTPB, ABCA3 and CDH1 in +CHIR and –CHIR cultures on day 49 of differentiation normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=1 differentiation round. (C) Representative immunofluorescence images of day 49 +CHIR and -CHIR cultures stained against SFTPB or E-Cadherin. Nuclei stained with DAPI. Scale bars 200µm.

41 Results

To further enhance differentiation towards ATII-like cells, the influence of Wnt withdrawal was investigated. A recent study described a positive effect of a temporal removal of Wnt signaling during late phases of hPSC-derived lung epithelial cell differentiation on the induction efficiency of distal alveolar epithelial lineages (Jacob et al., 2017). Therefore, CHIR99021 was removed from the culture medium between day 35 and day 42 of differentiation in iPSC line SFC084-03-01 (Figure 11A).

Figure 12. CD47+ MACS sorting of iPSC-derived lung epithelial progenitor cells. iPSC line SFC084-03-01. (A) Differentiation of iPSCs towards ATII-like cells with or without CD47+ MACS sorting of lung epithelial progenitor cells on day 24 of differentiation. RI = Y-27632; ActA = Activin A; SB = SB431542; Dor = Dorsomorphin; Nog = Noggin; RA = all-trans retinoic acid; CHIR = CHIR99021. DCI = dexamethasone, 8-Br- cAMP, IBMX. (B) Gene expression of NKX2.1, SOX9 and FOXA2 in day 32 cultures normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=1 differentiation round. Day 32 = replating as clumps on day 24. Day 32 unsorted = single-cell dissociation and re-seeding without sorting on day 24. Day 32 CD47+ = single-cell dissociation and CD47+ MACS sorting on day 24. Day 32 CD47- = single-cell dissociation and CD47- MACS sorting on day 24. (C) Gene expression of SFTPC, SFTPB and ABCA3 in day 49 cultures normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=1 differentiation round. Day 49 = replating as clumps on day 24. Day 49 CD47+ = single-cell dissociation and CD47+ MACS sorting on day 24. (D) Representative immunofluorescence images of day 49 and day 49 CD47+ cultures stained against SFTPB. Nuclei stained with DAPI. Scale bars 200µm.

42 Results

Preliminary qRT-PCR data showed that CHIR99021 withdrawal (-CHIR) resulted in higher expression levels of the ATII markers SFTPC, SFTPB and ABCA3 in day 49 cultures compared to controls (+CHIR), while no change in pan-epithelial CDH1 expression was observed (Figure 11B). Immunofluorescence of day 49 ALI cultures confirmed that temporal removal of CHIR99021 resulted in the formation of more SFTPB+ cells, while E- Cadherin was uniformly expressed in both +CHIR and –CHIR cultures (Figure 11C). These results suggest that temporal CHIR99021 withdrawal favors ATII-like cell differentiation in our iPSC-derived ALI model. Therefore, this step was incorporated into the final differentiation protocol.

It has recently been reported that hPSC-derived NKX2.1+ precursor cells are characterized by a CD47high cell surface phenotype that allows their prospective isolation without the need for genome editing and the usage of a reporter (Hawkins et al., 2017). Therefore, the effects of MACS sorting of CD47+ cells were tested in day 24 cultures derived from iPSC line SFC084-03-01 (Figure 12A). Preliminary data showed that by day 32 of differentiation, cultures sorted for CD47 on day 24 displayed a higher expression of SOX9 and FOXA2 than control cultures (dissociated and re-seeded without sorting), but NKX2.1 expression levels were only slightly affected (Figure 12B). Moreover, day 49 cultures derived from CD47+ cells displayed lower expression levels of the ATII markers SFTPC, SFTPB and ABCA3 compared to cultures differentiated according to the original protocol, in which day 24 progenitors are re-seeded as clumps, rather than as a single cell suspension (Figure 12C). No changes between Day 49 CD47+ cultures and Day 49 control cultures were observed by immunofluorescence, with both conditions containing multiple SFTPB+ cells that were mainly located on the apical culture surface (Figure 12D). As these preliminary data did not support a beneficial effect of CD47+ sorting, no sorting step was incorporated into the final differentiation protocol and further experiments were conducted with unsorted day 24 cultures that were seeded onto Transwell inserts as clumps.

3.3. Characterization of iPSC-derived lung epithelial progenitor cells

Figure 13. Optimized differentiation protocol to derive lung epithelial progenitor cells from iPSCs. (A) Step-wise differentiation of iPSCs towards lung epithelial progenitor cells. RI = Y-27632; ActA = Activin A; SB = SB431542; Dor = Dorsomorphin; Nog = Noggin; RA = all-trans retinoic acid; CHIR = CHIR99021. (B) Morphology of iPSC-derived cultures (iPSC line SFC065-03-03) at different stages of differentiation shown by light microscopy. Scale bars 100µm.

43 Results

Following the optimization of the protocol, lung epithelial progenitor cells derived from iPSC lines SFC065-03-03 and SFC084-03-01 were characterized in detail to confirm the robustness of the optimized differentiation protocol (Figure 13).

The relative expression levels of selected marker genes were measured at different timepoints during differentiation and revealed similar expression kinetics for both iPSC lines (Figure 14). Pluripotency marker OCT4 was expressed in iPSCs and silenced upon differentiation. DE markers SOX17 and CXCR4 were induced by day 4 of differentiation. FOXA2 expression peaked on day 4 of differentiation and was then reduced in day 7 AFE cultures, but continued being expressed throughout the differentiation towards lung epithelial progenitor cells. SOX2 was highly expressed in iPSCs and day 7 AFE cultures and NKX2.1 expression increased over time and displayed its highest expression levels in day 24 lung epithelial progenitor cells.

Figure 14. Changes in gene expression during iPSC towards lung epithelial progenitor cell differentiation. Gene expression of OCT4, SOX17, CXCR4, SOX2, FOXA2 and NKX2.1 from day 0 to day 24 of differentiation normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. (A) iPSC line SFC065-03-03. (B) iPSC line SFC084-03-01.

44 Results

Subsequently, an in depth characterization of lung epithelial progenitor cells derived from iPSC line SFC065-03-03 was performed.

By day 4 of differentiation, 91.3% (±4.9%) of nuclei were SOX17+ (Figure 15A). DE cells were differentiated towards anterior foregut endoderm cells, which then ventralized and expanded. Immunofluorescence analysis confirmed that these cells were triple-positive for NKX2.1, FOXA2 and SOX9, as characteristic of distal lung epithelial progenitor cells (Figure 15B). Flow cytometry analysis showed that the mean percentage of NKX2.1+ cells after 24 days of differentiation was 58.9% (±4.7%) (Figure 15C). The expression of NKX2.1, FOXA2, SOX9 and ID2 was significantly induced in day 24 cultures compared to day 0

Figure 15. Characterization of lung epithelial progenitor cells derived from iPSC line SFC065-03-03. (A) Representative immunofluorescence image of day 4 definitive endoderm (DE) cells. Mean percentage of SOX17+ nuclei (± SEM) from 3 independent experiments is indicated. Scale bar 100 µm. (B) Representative immunofluorescence image of day 24 lung progenitors stained against NKX2.1, FOXA2 and SOX9. Nuclei stained with Hoechst 33342. Scale bar 50µm. (C) Representative flow cytometry analysis of NKX2.1+ cells on day 24. Mean percentage of NKX2.1+ cells (± SEM) from 3 independent differentiation rounds is indicated. (D) Expression of selected marker genes on day 24 of differentiation normalized to GAPDH relative to day 0 (iPSCs). Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds for NKX2.1, FOXA2, SOX9, ID2 and OCT4 and from n=1 differentiation round for PAX6, PAX8, AFP and ALB. ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Mann-Whitney U-test. Hum. lung = total human lung. HepG2 = HepG2 hepatic cancer cell line. (E) Representative images of 3D branching organoids obtained by expanding lung epithelial progenitors in Matrigel from day 24 of differentiation onwards without switching to a terminal differentiation medium. White arrow heads = bifurcation points. Scale bars 1000 µm (day 49, left) and 400 µm (right). Image from Schruf et al., 2020.

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(Figure 15D). On the other hand, no induction of the neuro-ectodermal marker PAX6 or the thyroid marker PAX8 was observed. While a mild induction of the liver markers AFP and ALB was observed, relative expression levels remained multiple magnitudes below those present in HepG2 hepatocellular carcinoma cells and ALB expression levels in iPSC- derived day 24 cultures were comparable to those detected in total human lung. 3D Matrigel culture was utilized to confirm the budding and branching potential of the iPSC-derived lung epithelial progenitor cells, which constitutes a key functional feature of fetal epithelial progenitors during lung morphogenesis (Figure 15E).

A similar characterization was also conducted for lung epithelial progenitor cells derived from iPSC line SFC084-03-01. This line produced an average of 96.5% (±2.0%) SOX17+ day 4 DE cells and 53.9% (±2.4%) NKX2.1+ day 24 cells and the expression of further lung epithelial progenitor cell markers was detected on mRNA and protein level (Figure 16A-D).

Figure 16. Characterization of lung epithelial progenitor cells derived from iPSC line SFC084-03-01. (A) Representative immunofluorescence image of day 4 definitive endoderm (DE) cells. Mean percentage of SOX17+ nuclei (± SEM) from 3 independent experiments is indicated. Scale bar 100 µm. (B) Representative immunofluorescence image of day 24 lung progenitors stained against NKX2.1, FOXA2 and SOX9. Nuclei stained with Hoechst 33342. Scale bar 50µm. (C) Flow cytometry analysis of NKX2.1+ cells on day 24. Mean percentage of NKX2.1+ cells (± SEM) from 3 independent differentiation rounds is indicated. (D) Expression of selected marker genes on day 24 of differentiation normalized to GAPDH relative to day 0 (iPSCs). Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds for NKX2.1, FOXA2, SOX9, ID2 and OCT4 and from n=1 differentiation round for PAX6, PAX8, AFP and ALB. *** P < 0.001 and **** P < 0.0001 by Mann-Whitney U-test. Hum. lung = total human lung. HepG2 = HepG2 hepatic cancer cell line. Images (A), (B) and (C) from Schruf et al., 2020.

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3.4. Characterization of iPSC-derived ATII-like cells differentiated in ALI culture

Using the optimized differentiation protocol (Figure 17A), a detailed characterization of ATII-like cells derived from iPSC lines SFC065-03-03 or SFC084-03-01 was performed.

Figure 17. Differentiation of iPSCs towards ATII-like cells in ALI culture. iPSC line SFC065-03-03. (A) Optimized iPSC to ATII-like cell differentiation protocol. RI= Y-27632; ActA = Activin A; SB = SB431542; Nog = Noggin; RA = all-trans retinoic acid; CHIR = CHIR99021; DCI = dexamethasone, 8-Br-cAMP, IBMX. (B) Alveolar epithelial marker gene expression on day 49 of differentiation normalized to GAPDH relative to day 0. Day 49 sub. = submerged conditions. Day 49 = ALI from day 32 of differentiation onwards. Hum. lung = total human lung control. Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. ns not significant, **** P < 0.0001 by Mann-Whitney U-test. (C) Representative immunofluorescence image of a day 49 ALI culture cross-section stained against SFTPC. Nuclei stained with DAPI. Dashed line indicates PET membrane of Transwell insert. Scale bar 50 µm. Image from Schruf et al., 2020.

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To induce ATII-like differentiation, day 24 lung epithelial progenitor cells were seeded onto Transwell inserts and, from day 32 onwards, the confluent cultures were further differentiated at ALI until day 49. Day 49 cells (line SFC065-03-03) differentiated at ALI displayed significantly upregulated expression levels of the ATII markers SFTPC, SFTPB and ABCA3 compared to submerged conditions, while no induction of the ATI markers CAV1 or PDPN was observed under either condition (Figure 17B). Staining of cross- sectioned day 49 ALI cultures revealed that SFTPC+ cells resided mainly on the apical surface of the cultures (Figure 17C). These results show that air-exposure had a positive effect on ATII-like cell differentiation in the described model system.

Figure 18. Time-course of alveolar epithelial-associated transcript expression during the maturation of iPSC-derived ALI cultures. iPSC line SFC065-03-03. (A) Alveolar epithelial marker gene expression from day 24 to day 49 of differentiation normalized to GAPDH relative to day 0. ALI = start of air-liquid interface. Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. (B) Heatmap of top deregulated genes in day 49 vs. day 24 cultures by RNA-seq (cut-off: q-val < 0.001, |logFC| > 2, maxRPKM > 10) with known ATII marker genes labelled on the right. Day 0 = iPSCs. Day 24 = lung epithelial progenitors. Day 49 = ATII-like cells. Image from Schruf et al., 2020.

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An induction of SFTPC, SFTPB and ABCA3 expression was observed over time by qRT-PCR, while CAV1 expression was not induced (Figure 18A). Additionally, RNA-seq analysis revealed that many other known ATII associated transcripts (SFTPA2, SCD, CEACAM6, TM4SF1, NAPSA, CTSH, SFTPA1, PGC, SFTPD, LPCAT1, LAMP3, CEACAM5, GPR116 and SLC34A2) were upregulated in day 49 cultures (Figure 18B).

Day 49 cultures also showed upregulated expression of the human alveolar epithelial progenitor marker TM4SF1 compared to iPSCs. Moreover, a moderate induction of basal cell markers (KRT5, TP63) and other airway related transcripts (SCGB1A1, MUC5AC, MUC5B and FOXJ1) compared to day 0 was observed (Figure 19).

Immunofluorescence of day 49 cultures showed the homogeneous expression of the pan-epithelial marker E-Cadherin and confirmed the presence of SFTPC+, SFTPB+ and ABCA3+ cells, indicative of an ATII cell phenotype (Figure 20A). In line with the upregulation of basal cell marker expression on mRNA level, rare KRT+ cells could also be detected, indicating that in addition to ATII-like cells, our model system contains airway basal- like cells.

ATII-specific lamellar body-like structures were identified in day 49 cultures by transmission electron microscopy (Figure 20B). In accordance, live-stained iPSC-derived Figure 19. Expression of transcripts associated with ATII-like cells contained large alveolar and airway epithelial cell fates in day 49 organelles displaying intensive cultures derived from iPSC line SFC065-03-03. Gene expression in iPSC-derived cultures and in total human accumulation of LysoTracker Green lung normalized to GAPDH relative to day 0 (iPSCs). Day DND-26 (Figure 20C). Single cell 0 = iPSCs. Day 49 = iPSC-derived ATII-like cells. Hum. dissociation and replating of iPSC- lung = total human lung. Data are presented as mean fold change ± SEM from n=9 independent differentiation derived ATII-like cells under 2D rounds. Image from Schruf et al., 2020. submerged conditions in serum-

49 Results containing medium resulted in downregulated SFTPC, SFTPB and ABCA3 expression and upregulated expression of the ATI markers CAV1 and PDPN, indicative of ATI-like differentiation potential (Figure 20D, E).

Figure 20. ATII-like phenotypic and functional properties of ALI cultures derived from iPSC line SFC065- 03-03. (A) Representative immunofluorescence images of day 49 cultures stained against SFTPC, SFTPB, ABCA3, E-Cadherin or KRT5 (filled arrow head). Nuclei stained with DAPI. Scale bars 50µm. (B) Lamellar body- like structures in day 49 cultures identified by transmission electron microscopy. Scale bars in µm. (C) LysoTracker Green live-staining of iPSC-derived ATII-like cell. Nucleus stained with Hoechst 33342. Scale bar 10 µm. Representative image from n=3 independent differentiation rounds. (D) Morphology of day 54 cultures remaining in ALI culture on Transwell inserts (day 54) and day 54 cultures replated onto tissue culture-treated plastic on day 49 and cultured under submerged conditions in 10% FBS medium to induce ATI-like differentiation (day54 replated). Scale bars 50µm. (E) Gene expression of selected alveolar epithelial marker genes on day 54 of differentiation normalized to GAPDH relative to day 0 (iPSCs). Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. ** P < 0.01, **** P < 0.0001 by Mann-Whitney U-test. Images (A), (B), (C) and (E) from Schruf et al., 2020.

To confirm its reproducibility, the described differentiation protocol was also applied to iPSC line SFC084-03-01. Day 49 cultures derived from this line showed upregulated expression of ATII markers and a moderate induction of basal cell markers and other airway related transcripts compared to day 0 (Figure 21A). Moreover, immunofluorescence confirmed the presence of SFTPC+ and SFTPB+ cells and transmission electron microscopy revealed that

50 Results lamellar body-like structures were present within the cultures, confirming the ATII-like phenotype of day 49 cultures derived from the alternative iPSC line (Figure 21B, C). In summary, these data demonstrate the establishment of a robust directed differentiation protocol to derive SFTPC+ ATII-like cells from human iPSCs in ALI culture.

Figure 21. Expression of transcripts associated with alveolar and airway epithelial cell fates and ATII- like phenotype of day 49 cultures derived from iPSC line SFC084-03-01. (A) Gene expression in iPSC- derived cultures and in total human lung normalized to GAPDH relative to day 0 (iPSCs). Day 0 = iPSCs. Day 49 = iPSC-derived ATII-like cells. Hum. lung = total human lung. Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. (B) Representative immunofluorescence images of day 49 cultures stained against SFTPC or SFTPB. Nuclei stained with DAPI. Scale bars 50µm. (C) Lamellar body-like structures in day 49 cultures identified by transmission electron microscopy. Scale bars in µm. Images (A) and (B) from Schruf et al., 2020.

3.5. Design of a novel IPF-relevant cytokine cocktail to mimic aspects of a pro- fibrotic lung milieu in vitro

As it has been hypothesized that the exposure to a pro-fibrotic environment is a potential driver of epithelial dysfunction in IPF, this thesis aimed to incorporate fibrosis-related stimuli

51 Results into the established iPSC-derived system to model this aspect of IPF pathogenesis. Therefore, an IPF-relevant cytokine cocktail (IPF-RC) for the long-term stimulation of iPSC- derived cultures was designed to simulate the cytokine milieu present in an IPF lung. The composition of the IPF-RC, which is based on a literature research, is listed in detail in Table 2. Briefly, the IPF-RC contains nine cytokines previously shown to be upregulated in IPF bronchoalveolar lavage (BAL) or sputum samples compared to healthy controls (TGF- β1, IL-1β, TNF-α, IL-8, MCP1, IL-33, TSLP, IL-13 and IL-4). This cytokine cocktail was capable of inducing collagen I formation in primary human lung fibroblasts at different dilutions without exerting any cytotoxic effects (Figure 22). For all consecutive experiments, IPF-RC was utilized at a final dilution of 1:1000.

Figure 22. IPF-RC-induced collagen I formation in primary human lung fibroblasts. (A) Representative immunofluorescence images utilized for quantification. Nuclei stained with DAPI. Scale bars 100µm. Image based analysis of the number of (B) nuclei and (C) collagen I in primary human lung fibroblasts treated with IPF- RC at different dilutions for 72 h. Bars represent mean from n=3 independent experiments. Error bars represent SEM. * P < 0.05, **** P < 0.0001 by Mann-Whitney U-test. Image from Schruf et al., 2020.

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Table 2. Literature-based composition of an IPF-relevant cytokine cocktail (IPF-RC)

IPF-RC (final conc. at Cytokine Previous investigations of IPF BAL or sputum a dilution of 1:1000)

 32 pg/mL in IPF sputum (n=15), undetectable in healthy sputum (n=30) (Guiot et al., 2017)  22.1 pg/mL in IPF with emphysema BAL (n=38); 14.4 TGF-β1 0.3 ng/ mL pg/mL in IPF without emphysema BAL (n=64) (Tasaka et al., 2012)  1500 pg/mL in IPF BAL (n=16) (Zhang et al., 2016)  0.6 pg/mL in IPF BAL (n=77), <0.3 pg/mL in healthy IL-1β BAL (n=349) (Barlo et al., 2011) 0.01 ng/ mL  2.4 pg/mL in IPF BAL (n=11) (Bellanger et al., 2016)  1.9 pg/mL in IPF BAL (n=11) (Bellanger et al., 2016)  10 pg/mL in IPF sputum (n=15), 6.8 pg/mL in healthy sputum (n=30) (Guiot et al., 2017) TNF-α 0.1 ng/ mL  0.87 pg/mL in IPF with emphysema BAL (n=38); 0.41 pg/mL in IPF without emphysema BAL (n=64) (Tasaka et al., 2012)  38.4 pg/mL in IPF BAL (n=11) (Bellanger et al., 2016)  276 pg/mL in IPF sputum (n=15), 46 pg/mL in healthy sputum (n=30) (Guiot et al., 2017)  35 pg/mL in IPF BAL (n=13), 15 pg/mL in healthy BAL IL-8 (n=9) (Meloni et al., 2004) 1.5 ng/ mL  179 pg/mL in IPF with emphysema BAL (n=38); 108 pg/mL in IPF without emphysema BAL (n=64) (Tasaka et al., 2012)  44.6 pg/mL in IPF BAL (n=7) (Vasakova et al., 2009)  70 pg/mL in IPF BAL (n=13), 5 pg/mL in healthy BAL (n=9) (Meloni et al., 2004) MCP1  555 pg/mL in IPF with emphysema BAL (n=38); 351 0.7 ng/ mL pg/mL in IPF without emphysema BAL (n=64) (Tasaka et al., 2012)  4.13 pg/mL in IPF BAL (n=100), 1.15 pg/mL in healthy IL-33 0.04 ng/ mL BAL (n=40) (Lee et al., 2017)  2.5 pg/mL in IPF BAL (n=11) (Bellanger et al., 2016) TSLP  9.27 pg/mL in IPF BAL (n=100), 4.49 pg/mL in healthy 0.1 ng/ mL BAL (n=40) (Lee et al., 2017)  250 pg/mL in IPF BAL (n=16), 60 pg/mL in healthy BAL IL-13 2.5 ng/ mL (n=8) (Park et al., 2009)  16 pg/mL in IPF BAL (n=16), 3 pg/mL in healthy BAL IL-4 0.16 ng/ mL (n=8) (Park et al., 2009)

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3.6. IPF-RC stimulation of iPSC-derived distal lung epithelial progenitor cells induces an IPF-related phenotype and shifts differentiation towards proximal lineages

To investigate the effects of a pro-fibrotic environment on ATII cell differentiation, iPSC- derived alveolar epithelial progenitor cells were stimulated with IPF-RC for two weeks following induction of SFTPC expression (Figure 18A; Figure 23A). All experiments described in the following paragraphs were performed in iPSC line SFC065-03-03.

Figure 23. Influence of IPF-RC stimulation on MMP secretion from iPSC-derived lung epithelial cultures. iPSC line SFC065-03-03. (A) Schematic for IPF-RC stimulation during alveolar epithelial differentiation (day 35 - day 49). (B) Total cell number per Transwell insert. Day 49 = iPSC-derived ATII-like cells (control). Day 49 +IPF-RC = iPSC-derived cells treated with IPF-RC from day 35 of differentiation onwards. Data are presented as mean ± SEM from n=4 independent differentiation rounds. ns not significant by Mann-Whitney U-test. (C) Caspase-3/ 7 activity. Data are presented as mean ± SEM from n=3 independent differentiation rounds. ns not significant by Mann-Whitney U-test. (D) MMP-7 and MMP-10 protein levels in culture medium measured by ELISA. Data are presented as mean ± SEM from n=3 independent differentiation rounds. * P < 0.05, ** P < 0.01 by Mann-Whitney U-test. Image from Schruf et al., 2020.

No changes in total cell number or caspase-3/7 activity were detected upon IPF-RC treatment, suggesting that IPF-RC did not exert any anti-proliferative or pro-apoptotic effects (Figure 23B, C). qRT-PCR revealed an upregulated expression of the IPF-related marker genes MMP7, MMP10 and BPIFB1, while the expression of CDH1, ACTA2 and COL1A1 was not altered (Figure 24). In addition, significantly increased MMP-7 and MMP- 10 protein levels were detected in the culture medium of day 49 cultures stimulated with IPF-RC by ELISA (Figure 23D).

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Figure 24. Effect of IPF-RC treatment on the expression of transcripts known to be deregulated in IPF lungs in iPSC-derived ATII-like cells. iPSC line SFC065-03-03. qRT-PCR showing expression normalized to GAPDH relative to day 0 (iPSCs). Bars represent mean fold changes from n=6 independent differentiation rounds. Error bars represent SEM. ns not significant, * P < 0.05, ** P < 0.01 and *** P < 0.001 by two-tailed unpaired Student’s t-test. Image from Schruf et al., 2020.

RNA-seq was performed to further investigate IPF-RC mediated transcriptional changes (Figure 25A). In accordance with an upregulation of MMP-7 and MMP-10 secretion, gene set enrichment analysis revealed a significant enrichment of transcripts associated with extracellular matrix organization (among top 10 associated gene sets under investigation; C2.CP.REACTOME (Curated)) for the day 49 +IPF-RC phenotype (Figure 25B). The expression data from the iPSC-derived model were also compared to a publicly available dataset for human IPF and healthy lung tissue (Kusko et al., 2016). Calculation of the intersection of these data sets revealed a significant overlap of both downregulated (p = 4.32x10-16) and upregulated (p = 2.5x10-29) transcripts between day 49 cultures treated with IPF-RC and human IPF lungs (Figure 25C). Epithelial cell adhesion-related transcripts (GJB1, TJP2, PCDH1 and OCLN), as well as transcripts that are known to be repressed in IPF, including KLF4, VEGFA, IL32 and SLC19A3, were among the downregulated genes. MMP10 and the airway epithelial associated transcripts BPIFB1, SOX2, PAX9, TP63, FOXJ1, SCGB1A1 and KRT5, were upregulated in both datasets. These results indicate that IPF-RC stimulation of our iPSC-derived ALI model system allows for the recapitulation of certain IPF-relevant processes and transcriptional changes, in vitro.

As RNA-seq revealed that known airway epithelial-related transcripts were among the upregulated transcripts upon IPF-RC stimulation, it was sought to determine if treatment with IPF-RC had the potential to alter the proximo-distal differentiation pattern of iPSC- derived lung epithelial progenitors. qRT-PCR analysis at different timepoints during differentiation revealed elevated expression levels of the transcription factor SOX2 and

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Figure 25. Fibrosis-related transcriptional changes induced by IPF-RC stimulation of iPSC-derived lung epithelial progenitors. iPSC line SFC065-03-03. (A) Principal component analysis of RNA-seq data representing the top 5000 transcripts according to the coefficient of variation. Arrows represent experimental timecourse. (B) Enrichment plot for the extracellular matrix organization gene set from Reactome. FDR q-value = 0.039. (C) Intersection between deregulated transcripts in IPF-RC-treated iPSC-derived day 49 cultures (vs. untreated) and deregulated transcripts in human IPF lung tissue (vs. healthy). Cut-off: q-val < 0.05, |logFC| > 0.5, maxRPKM > 2. Top section represents down-regulated, bottom section up-regulated transcripts. Boxes contain examples of commonly up- or down-regulated transcripts. Image from Schruf et al., 2020.

reduced expression of SOX9 upon IPF-RC stimulation, both of which are central players in proximo-distal epithelial patterning during lung development (Figure 26A). IPF-RC-treated day 49 cultures displayed significantly reduced expression of ATII-specific SFTPC, while the airway-related transcripts SCGB1A1, MUC5B, FOXJ1, KRT5 and TP63, which are also expressed in the abnormal epithelium lining cystic lesions found in distal IPF lungs, were significantly upregulated (Figure 26B). In contrast to MUC5B upregulation, expression of MUC5AC remained unchanged upon IPF-RC treatment, indicating that goblet cell- associated mucin induction was specific to MUC5B, similar to the bronchiolized epithelium in IPF patients. Expression of CAV1 and PDPN was not altered, indicating that differentiation of ATII-like cells towards ATI-like cells did not contribute to the loss of SFTPC expression.

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Figure 26. IPF-RC-induced distal-to-proximal shift in gene expression of iPSC-derived lung epithelial cultures. iPSC line SFC065-03-03. (A) SOX2 and SOX9 expression from day 24 to day 49 of differentiation normalized to GAPDH relative to day 0. ALI = start of air-liquid interface. ±IPF-RC = start of IPF-RC/ vehicle treatment. Data are presented as mean fold change ± SEM from n=3 independent differentiation rounds. (B) Alveolar and airway epithelial marker expression in day 49 cultures ±IPF-RC treatment normalized to GAPDH relative to day 0. Data are presented as mean fold change ± SEM from n=9 independent differentiation rounds. ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by two-tailed Student’s t-test. Image from Schruf et al., 2020.

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In accordance with the reduced SFTPC expression in IPF-RC cultures, the mean surface area covered by SFTPC+ cells was significantly reduced in day 49 IPF-RC cultures compared to controls, indicating a loss of ATII-like cells (Figure 27A, B). Histological analysis of cross-sectioned day 49 cultures revealed that IPF-RC stimulation resulted in the emergence of Alcian Blue/ PAS+ cell clusters, displaying a secretory cell-like morphology (Figure 27C). Moreover, areas of MUC5B+ goblet-like cells, which were absent in controls, were detected in IPF-RC cultures by immunofluorescence (Figure 27D). These results show that IPF-RC led to the emergence of MUC5B+ secretory cells, which are also frequently observed lining cystic lesions in the distal lung of IPF patients, at the expense of ATII-like cell differentiation.

Figure 27. Epithelial phenotypic changes recapitulating aspects of IPF-related bronchiolization resulting from IPF-RC stimulation during differentiation. iPSC line SFC065-03-03. (A) Immunofluorescence against SFTPC in day 49 cultures ±IPF-RC. Nuclei stained with DAPI. Scale bars 50µm. (B) SFTPC+ surface area quantified by immunofluorescence. Mean SFTPC+ area per image ± SEM from 3 indipendent differentiation rounds (with 3 biological replicates per experiment and 9 images per replicate). * P < 0.05 by Mann-Whitney U- test. (C) Representative H&E and Alcian Blue/ Periodic acid-Schiff (PAS) stainings of iPSC-derived culture cross-sections. Scale bars 50 µm. (D) Immunofluorescence against MUC5B in day 49 cultures ±IPF-RC. Nuclei stained with DAPI. Scale bars 40µm. Image from Schruf et al., 2020.

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To understand the relative contribution of mediators within the IPF-RC that are known to induce epithelial remodeling (IL-13, TGF-β1 or TNF-α), individual components were removed from the cocktail. Day 49 cultures differentiated in the presence of IPF-RC without IL-13 expressed higher levels of SFTPC than cells treated with the full cytokine cocktail and

Figure 28. Alterations in the effect of IPF-RC on iPSC-derived ATII-like cell differentiation following removal of single cytokines from the cocktail. Gene expression of IPF-related, alveolar and airway epithelial marker genes in iPSC-derived ATII-like cells normalized to GAPDH relative to day 0 (iPSCs). Removal of single cytokines from the cytokine cocktail. Day 49 = iPSC-derived ATII-like cells (control). Day 49 +IPF-RC = iPSC- derived cells treated with IPF-RC from day 35 of differentiation onwards. Day 49 +IPF-RC w/o IL-13 = iPSC- derived cells treated with IPF-RC without addition of IL-13 from day 35 of differentiation onwards. Day 49 +IPF- RC w/o TGF-β1 = iPSC-derived cells treated with IPF-RC without addition of TGF-β1 from day 35 of differentiation onwards. Day 49 +IPF-RC w/o TNF-α = iPSC-derived cells treated with IPF-RC without addition of TNF-α from day 35 of differentiation onwards. Bars represent mean fold changes from n=3 independent differentiation rounds. Error bars represent SEM. ns not significant, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by two-way ANOVA and Tukey test. Image from Schruf et al., 2020.

59 Results displayed lower expression of MMP10, BPIFB1, MUC5B, FOXJ1 and TP63, suggesting IL- 13 as a main driver of the IPF-related effects exerted by IPF-RC (Figure 28). However, removal of IL-13 did not reduce the expression of MMP7, SCGB1A1 and KRT5 compared to the full cocktail. Furthermore, stimulation with 2.5 ng/mL IL-13 alone resulted in a significant downregulation of SFTPC expression and upregulation of FOXJ1 and TP63 expression, but did not alter the expression of MMP10, BPIFB1 or MUC5B, indicating that IL-13 was not solely responsible for the proximalization effect of IPF-RC (Figure 29). IPF- RC without TGF-β1 led to a reduction in MUC5B expression compared to the full cocktail,

Figure 29. Influence of IL-13 stimulation on iPSC-derived ATII-like cell differentiation. Gene expression of IPF-related, alveolar and airway epithelial marker genes in iPSC-derived ATII-like cells normalized to GAPDH relative to day 0 (iPSCs). Stimulation with IL-13 only at the same concentration as included in IPF-RC. Day 49 = iPSC-derived ATII-like cells (control). Day 49 +IL-13 = iPSC-derived cells treated with 2.5 ng/ mL IL-13 from day 35 of differentiation onwards. Bars represent mean fold changes from n=3 independent differentiation rounds. Error bars represent SEM. * P < 0.05, *** P < 0.001 and **** P < 0.0001 by Mann-Whitney U-test. Image from Schruf et al., 2020.

yet failed to reduce IPF-RC mediated expressional changes in any of the other transcripts analyzed (Figure 28). Removal of TNF-α did not result in a reduced induction of any IPF- or airway-associated transcripts compared to IPF-RC (Figure 28). Moreover, TGF-β1 or

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TNF-α removal failed to rescue SFTPC expression (Figure 28). These results indicate that TGF-β1 and TNF-α were not the main drivers of the bronchiolization-like effect cause by IPF-RC in our model system.

Figure 30. Effects of nintedanib treatment on the transcriptional changes induced by IPF-RC stimulation of iPSC-derived ATII-like cells. Gene expression of alveolar, airway epithelial and IPF-related marker genes in iPSC-derived ATII-like cells and normalized to GAPDH relative to day 0 (iPSCs). Day 49 = iPSC-derived ATII- like cells (control). Day 49 +IPF-RC = iPSC-derived cells treated with IPF-RC from day 35 of differentiation onwards. Day 49 +100 nM nintedanib = iPSC-derived cells treated with nintedanib from day 35 of differentiation onwards. Day 49 +IPF-RC +100 nM nintedanib = iPSC-derived cells treated with IPF-RC and nintedanib from day 35 of differentiation onwards. Bars represent mean fold changes from n=3 independent differentiation rounds. Error bars represent SEM. ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by two-way ANOVA and Tukey test. Image from Schruf et al., 2020.

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Nintedanib, a tyrosine kinase inhibitor, is one of only two currently approved pharmacological agents for the treatment of IPF and has been shown to slow down IPF- related lung function decline, but its effect on the alveolar epithelium is not completely understood. In the iPSC-derived model system established in this thesis, nintedanib only showed some minor effects on SCGB1A1 and TP63 expression at clinically relevant concentrations (100 nM), but was neither able to rescue induction of IPF- and other airway- related marker genes, nor the reduction in SFTPC expression in IPF-RC-treated cells (Figure 30). These results suggest that the mechanisms underlying the bronchiolization- like effects observed in our model system are not directly targeted by nintedanib.

3.7. IPF-RC treatment of primary small airway basal cells favors MUC5AC+ goblet cell differentiation and impairs ciliation

Airway basal cells have been proposed as a potential source of the bronchiolized distal lung epithelium in IPF. Therefore, the effect of IPF-RC treatment on primary small airway basal cell differentiation at air-liquid interface was assessed.

Figure 31. IPF-relevant cytokine cocktail (IPF-RC)-induced changes in gene expression in primary human SAEC cultures. Gene expression in primary human SAEC cultures normalized to GAPDH relative to untreated controls. Day 28 = SAECs differentiated for 28 days at air-liquid interface. Day 28 +IPF-RC = SAECs differentiated for 28 days at air-liquid interface in the presence of IPF-RC. Bars represent mean fold changes from n=3 independent experiments. Error bars represent SEM. ns not significant, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Mann-Whitney U-test. Image from Schruf et al., 2020.

SAEC cultures were stimulated with IPF-RC during air-liquid interface differentiation and transcriptional as well as phenotypic changes were assessed. Similar to iPSC-derived cultures, IPF-RC stimulation induced upregulated mRNA expression of KRT5, TP63, MMP7, MMP10 and BPIFB1, as well as increased secretion of MMP-7 and MMP-10 in SAEC cultures (Figure 31, Figure 32A-B). Conversely, expression of SCGB1A1 and FOXJ1 decreased compared to control cultures. While MUC5AC expression was enhanced, no

62 Results significant deregulation of MUC5B expression, the upregulation of which has been linked to epithelial dysfunction in IPF, could be detected. Moreover, cilia formation and ciliary function were severely impaired in IPF-RC-treated SAECs (Figure 32C). These results indicate that the effect of IPF-RC on primary small airway basal cell differentiation differs from its effect on iPSC-derived alveolar epithelial progenitor cell differentiation.

Figure 32. IPF-relevant cytokine cocktail (IPF-RC)-induced phenotypic and functional alterations in SAEC cultures derived from primary human small airway basal cells. (A) Representative hematoxylin and eosin staining (H&E, left), Alcian Blue/ Periodic acid-Schiff (PAS) staining (middle) and immunofluorescence double staining (right) of cross-sections of primary human SAEC cultures. Immunofluorescence against MUC5AC (green) and KRT5 (red). Nuclei stained with DAPI (blue). Scale bars 50µm. (B) MMP-7 and MMP-10 protein levels in cell culture supernatant of SAEC cultures. Bars represent mean concentration from n=3 independent experiments. Error bars represent SEM. ** P < 0.01, and **** P < 0.0001 by Mann-Whitney U-test. (C) Ciliary beat measurement in SAEC cultures. Left: Mean area covered by motile cilia and mean ciliary beat frequency calculated from n=3 independent experiments. 6 regions per insert from 3 biological replicates were analyzed for each experiment. Error bars represent SEM. **** P < 0.0001 by Mann-Whitney U-test. Right: Representative regions analyzed for cilia beat measurement. Ciliary movement recording for 6 seconds per region. Image from Schruf et al., 2020.

3.8. The metaplastic epithelium in distal IPF lungs contains atypical transition zones between SFTPC+ ATII-like cells and MUC5B+ goblet-like cells

To verify the relevance of the findings observed in our model system for human disease, immunofluorescence on IPF patient lung sections was performed. The heterogeneous cystic lesions found in the dense fibrotic tissue of the distal IPF lungs were lined by a simple single-layered columnar or cuboidal epithelium, interrupted by areas of epithelial denudation and areas of multi-layered hyperplastic epithelium. Double immunofluorescence against SFTPC and MUC5B was performed in three IPF lungs. In accordance with previous reports that identified MUC5B+ secretory cells as the predominant mucus cell type in the honeycomb epithelium, many of the cystic lesions found in the distal regions of IPF lungs were lined by MUC5B expressing columnar epithelial cells and displayed extensive mucus plugging. Immunofluorescence further confirmed the presence of SFTPC+ cells in distal IPF

63 Results patients’ lungs. Some cystic lesions were lined exclusively by a simple SFTPC+ epithelial cell layer, indicative of ATII cell hyperplasia (Figure 33). The luminal space of some lesions contained yellow appearing material, suggestive of a mixture of mucus and surfactant secretions. Interestingly, cystic lesions that were lined by a metaplastic epithelium, containing atypical transitions from a SFTPC+ epithelium to a MUC5B+ epithelium, were also detected in all analyzed IPF samples (Figure 33). These transition zones of ATII-like cells residing in close proximity to goblet-like cells are indicative of a loss of regional epithelial specification and may represent areas of progressing alveolar bronchiolization.

Figure 33. Epithelial transition zones in cystic lesions of IPF patient lungs. Representative hematoxylin and eosin staining (H&E) and immunofluorescence against SFTPC (green) and MUC5B (red) in three human IPF lungs. Epithelial transition zones in cystic lesions lined by SFTPC+ cells (filled arrowheads) and MUC5B+ cells (unfilled arrowheads). Nuclei stained with DAPI (blue). Scale bars 350µm in H&E images; 50µm in immunofluorescence images. Image from Schruf et al., 2020.

64 Discussion

4. DISCUSSION

The inability to access, isolate and maintain functional primary ATII cells in long-term culture has significantly hindered the efforts to study and understand the alveolar epithelium, and its role in disease. In recent years, directed differentiation of hPSCs has emerged as an alternative approach that allows for the de novo generation of specific human cell types. In the first part of this thesis, directed differentiation of human iPSCs was applied to establish a novel model that allows for ATII-like cell differentiation in ALI culture. This model mimics the physiological environment of the lung epithelium and provides easy accessibility of the cells.

In the second part of this work, this system was utilized to investigate alveolar epithelial dysfunction in IPF, a fatal lung disease. As recent reports have indicated that the pro-fibrotic cytokine milieu within the lung plays a critical role in the pathogenesis of IPF, the model was stimulated with an IPF-relevant cytokine cocktail. Stimulation of iPSC-derived alveolar epithelial progenitor cells with this cocktail increased IPF biomarker secretion, induced IPF- associated transcriptional changes, and impaired ATII cell differentiation. This work thereby describes the establishment of a human model system that recapitulates key aspects of IPF-related alveolar epithelial dysfunction in vitro. In conclusion, this project has led to the generation of a novel disease relevant platform for both basic research and drug discovery applications, demonstrating the utility of iPSC-derived systems to study complex pathophysiological processes in the human lung.

4.1. Establishment of an iPSC-derived ALI model system of alveolar epithelial differentiation

4.1.1. Derivation of NKX2.1+ lung epithelial progenitor cells from human iPSCs

Initially, three healthy human iPSC lines, SFC065-03-03, SFC084-03-01 and SFC086-03- 01, which were reprogrammed from skin fibroblasts via non-integrating Sendai virus, were acquired and quality control was performed. Chromosomal integrity was confirmed and all iPSC lines stained positively for OCT4, SOX2 and Nanog, key transcriptional regulators that are highly expressed in pluripotent cells, and for SSEA4, TRA-1-81 and TRA-1-60, cell surface markers of pluripotent stem cells (Draper et al., 2002; Henderson et al., 2002; Looijenga et al., 2003; Pan and Thomson, 2007; Pesce and Scholer, 2001). In addition, all iPSC lines were capable of forming endodermal, mesodermal and ectodermal derivates, confirming their differentiation potential towards all three germ layers (Thomson et al., 1998). These findings confirm that the three iPSC lines utilized in this thesis display characteristic morphological and functional features of pluripotency, making them suitable for directed differentiation experiments.

Subsequently, step-wise optimization of a previously published protocol was performed for the iPSC lines to differentiate them towards lung epithelial lineages by applying cell line- dependent modifications (Huang et al., 2015). Initial experiments demonstrated that all three iPSC lines were capable of endoderm commitment, as evidenced by the induction of SOX17 and FOXA2 expression, and confirmed that iPSC-derived 3D DE cultures could be dissociated and re-plated for further differentiation towards AFE (Zorn and Wells, 2009). For AFE differentiation, the two BMP antagonists Dorsomorphin dihydrochloride and Noggin were compared. Based on previous studies that showed the reemergence of the

65 Discussion pluripotency marker SOX2 as a foregut marker during endodermal development, Noggin was chosen for all consecutive experiments, as SOX2 expression was upregulated in Noggin-treated day 7 AFE cultures compared to DE cultures, while Dorsomorphin dihydrochloride failed to upregulate SOX2 expression (Green et al., 2011). The mechanisms leading to the different effects of the endogenous BMP antagonist Noggin and the small molecule inhibitor Dorsomorphin have not been finally clarified. One possibility is that the insufficient capability of Dorsomorphin to induce AFE stems from its known off- target effects on other kinases, including AMPK, VEGFR2, and PDGFR (Lowery et al., 2016). It has been described that close monitoring of seeding density and correct timing of AFE patterning are essential to achieve a sufficient induction efficiency of lung epithelial progenitor cells from hPSCs (Huang et al., 2015; Miller et al., 2019). In line with this, the different re-seeding densities and AFE induction time-points compared in this study resulted in varying induction efficiencies. The protocol that produced the highest yield of lung epithelial progenitor cells for all three iPSC lines was chosen for all further experiments.

In-depth characterization of lung epithelial progenitor cells derived from iPSC lines SFC065- 03-03 and SFC084-03-01 utilizing the optimized differentiation protocol was performed. The analysis of the expression kinetics of various transcription factors over time revealed that the relative expression of OCT4, SOX17, FOXA2, SOX2 and NKX2.1 compatibly changed over time as previously described for hPSC-derived lung epithelial progenitor cell differentiation (Gotoh et al., 2014; Green et al., 2011).

As accumulating evidence suggests that all mammalian lung epithelial cells derive from embryonic NKX2.1+ progenitors, NKX2.1+ cells were quantified by flow cytometry (Hawkins et al., 2017). After 24 days of differentiation, iPSC-derived cultures contained 58.9% (±4.7%) NKX2.1+ cells (SFC065-03-03) and 53.9% (±2.4%) NKX2.1+ cells (SFC084-03-01), respectively. These induction efficiencies are within the range of ~30% to ~85% that has previously been achieved utilizing similar sorting-free directed differentiation approaches across various different hPSC lines (Firth et al., 2014; Gotoh et al., 2014; Huang et al., 2014; Wong et al., 2012). NKX2.1 is also expressed during thyroid morphogenesis and in certain regions of the fetal brain (Lazzaro et al., 1991). Yet, day 24 cultures did not display expression of human neuroectoderm cell fate determinant PAX6, or PAX8, a marker of thyroid development (Gillam and Kopp, 2001; Zhang et al., 2010). However, moderate expression of the early liver markers AFP and ALB was detectable on day 24, indicative of a certain degree of heterogeneity within the derived endodermal progenitor cultures (Ikonomou and Kotton, 2015; Terrace et al., 2007).

Immunofluorescence of day 24 cells derived from iPSC lines SFC065-03-03 and SFC084- 03-01 showed that NKX2.1+ cells were also FOXA2+/SOX9+, and an induction of ID2 expression was observed by qRT-PCR. The expression signature of iPSC-derived day 24 cultures thereby corresponds to the one found in distal tip epithelial progenitors in fetal human lung during development (Nikolic et al., 2017). Importantly, when these iPSC- derived cells were cultured in Matrigel, they displayed three-dimensional branching, similar to primary human embryonic lung epithelial tip progenitors and lung epithelial progenitor cells derived from RUES2 embryonic stem cells (Chen et al., 2017; Nikolic et al., 2017). This demonstrates the capability of iPSC-derived lung epithelial progenitors to recapitulate an important functional aspect of lung development, which follows a defined branching program to build the continuous segments with multiple lateral branches that form the adult lung (Metzger et al., 2008).

66 Discussion

In order to increase the percentage of NKX2.1+ cells, efforts were made by other groups to identify cell surface markers that could distinguish NKX2.1+ and NKX2.1− cells to enable a purification of putative lung epithelial progenitor cells. A recent study found that a selection for CD47high/CD26low cells via FACS allows for an enrichment of hPSC-derived NKX2.1+ cells to ~90% (Hawkins et al., 2017). It was shown by the same group that these cells were capable of differentiating into alveolar epithelial cells and airway epithelial lineages (Jacob et al., 2017; McCauley et al., 2018a; McCauley et al., 2018b). Due to the subsequent observation that there was no significant difference in purification efficiency between combined CD47high/CD26low sorting and sorting for CD47high alone in six hPSC lines, the effect of CD47+ sorting of iPSC-derived day 24 cultures via MACS was assessed (Korogi et al., 2019). In our hands, CD47+ sorting of SFC084-03-01 day 24 cultures led to a moderate increase in NKX2.1 expression, but did not result in an increase of SFTPC, SFTPB or ABCA3 expression in day 49 cultures compared to controls. The choice therefore fell to the original sorting-free protocol, in which lung epithelial progenitors are re-seeded as clumps rather than single cells. The lack of a beneficial effect of CD47+ sorting might be explained by the fact that hPSC-derived lung epithelial progenitor cells require close cell-to-cell contact for survival. It has previously been observed that dissociation of VAFE into single cells can compromise viability and promote the outgrowth of non-lung lineages compared to clump passaging (Huang et al., 2015). Moreover, there is a possibility that the implementation of a single-cell dissociation step is not compatible with a subsequent differentiation in 2D monoculture, which has previously been shown to be prone to cell detachment (Huang et al., 2015). As a result, other groups have proceeded to culturing sorted lung epithelial progenitors in a 3D matrix with or without the addition of mesenchymal cells to achieve further differentiation into mature alveolar lineages (Chen et al., 2017; Yamamoto et al., 2017). In this thesis, alveolar maturation in an organoid format was not attempted, as the aim was to develop a 2D ALI model that mimics the physiological environment of lung epithelial cells and allows for easy accessibility of the cells’ apical surface. An alternative method that has been described for lung epithelial progenitor enrichment is sorting of cells highly positive for carboxypeptidase M (CPMhigh), the potential of which to differentiate into both alveolar and proximal airway lineages has been shown (Gotoh et al., 2014; Konishi et al., 2016; Yamamoto et al., 2017). A recent study compared the NKX2.1+ cell enrichment efficiency of CPM-based sorting and CD47-based methods and found CPM-based sorting to be more efficient in the six analyzed hPSC lines (Korogi et al., 2019). Therefore, an incorporation of this sorting method into our protocol should be considered in future experiments aiming to optimize lung progenitor induction efficiency. Alternatively, a higher progenitor yield could be achieved by applying genome editing to introduce a GFP reporter construct into the endogenous NKX2.1 locus of the respective iPSC lines which would allow for a GFP-based sorting of NKX2.1+ cells, as demonstrated previously (Hawkins et al., 2017; Longmire et al., 2012). However, these reporter-based approaches have been criticized for not being universally applicable, varying in faithfulness of reflecting gene expression between lines and for their potential to lead to artifacts (de Carvalho et al., 2019; Nikolic et al., 2018). In addition, the usage of reporter lines would not resolve the issue of single-cell dissociation discussed above.

Despite the achievement of high percentages of NKX2.1+ cells utilizing the aforementioned methods, the exact mechanisms driving the specification of individual lineages remain incompletely understood and the derivation of homogeneous populations of specific mature lung epithelial cell types remains challenging. For instance, it has recently been shown by single-cell RNA-seq that lung epithelial cultures derived from a pure population of sorted

67 Discussion

NKX2.1+ progenitors also contained hepatic and gastric progenies of unknown origin (McCauley et al., 2018a). In addition, it is unclear why different hPSC lines display such extensive variability in terms of their differentiation competence (Hawkins et al., 2017). It might therefore be useful to revert to sorting-free methods permissive for all mature lung epithelial cells to study lineage relationships in the developing and adult lung and to uncover the mechanisms underlying proximo-distal specification (de Carvalho et al., 2019). This knowledge could then be applied to develop more suitable differentiation regimens and novel sorting strategies.

4.1.2. Maturation of iPSC-derived lung epithelial progenitors towards ATII-like cells

Following the induction of NKX2.1+ lung epithelial progenitor cells, the next objective was further differentiation into alveolar lineages, mainly the maturation into SFTPC+ ATII cells. As preliminary data suggested that iPSC line SFC086-03-01 could not produce SFTPC+ cells under the tested culture conditions, this line was excluded from further experiments. In contrast, SFTPC expression was induced in iPSC lines SFC065-03-03 and SFC084-03- 01 utilizing an identical directed differentiation approach. The variable ATII-like cell differentiation capacity that was observed is in accordance with previous reports describing an extensive variability across hPSC lines in terms of their differentiation competence towards specific lineages (Bock et al., 2011; Hawkins et al., 2017). It has been hypothesized that the variation in differentiation potential between different iPSC lineages may result from donor-dependent as well as original cell type-dependent differences in genetic background or epigenetic landscape (Kim et al., 2011; Rouhani et al., 2014).

In the course of the optimization of the differentiation protocol utilized in this work, the effects of removing the GSK-3 inhibitor CHIR99021 from the culture medium between day 35 and day 42 of differentiation was investigated. Wnt signaling is known to play an essential role in distal mouse lung morphogenesis by regulating proximo-distal differentiation and epithelial self-renewal during alveogenesis as well as the maintenance of alveolar stem cells in the adult lung (Frank et al., 2016; Li et al., 2002; Nabhan et al., 2018; Shu et al., 2005). It has further been shown that early withdrawal of Wnt signaling from hPSC-derived lung epithelial progenitors prompts rapid proximal patterning, resulting in differentiated airway organoids containing secretory, multiciliated and basal cells (McCauley et al., 2017). On the other hand, temporal exposure to low Wnt conditions during late phases of hPSC-derived lung epithelial cell differentiation had a positive effect on the induction efficiency of distal alveolar epithelial lineages (Jacob et al., 2017). A different group reported that a continued presence of CHIR99021 following lung epithelial progenitor induction promoted cellular expansion and inhibited multilineage differentiation in hPSC-derived lung organoids (de Carvalho et al., 2019). In line with the observations made by Jacob et al., withdrawal of CHIR99021 for one week during late differentiation resulted in higher expression of ATII markers in our model system, confirming the beneficial effect of this temporal modulation of Wnt signaling on alveolar differentiation. Taken together, these findings and previous studies utilizing hPSCs implicate that a tight regulation of Wnt signaling is required for normal proximo-distal patterning of the developing lung epithelium (Nikolic et al., 2018).

This work aimed to adapt previously described differentiation approaches to derive alveolar epithelial cells from iPSCs in ALI culture. In addition to their physiological relevance for lung cells, ALI cultures provide the great advantage of easy accessibility to the epithelium’s apical surface and their resulting applicability to assessing the efficacy of inhaled drugs as well as the toxicity of aerosols or cigarette smoke (Latvala et al., 2016; Loret et al., 2016;

68 Discussion

Movia et al., 2018). While ALI culture has been applied to hPSC-derived airway epithelial cells to enhance maturation, this method has not yet been tested in hPSC-derived models of the alveolar epithelium (Firth et al., 2014; Wong et al., 2012). Our data show that iPSC- derived lung epithelial progenitor cells differentiated at ALI expressed higher levels of SFTPC, SFTPB and ABCA3 compared to submerged controls. This is in line with previous observations by Dobbs et al. and Alcorn et al. who found that ALI culture of primary human ATII cells has a beneficial effect on the maintenance of their cellular characteristics, including cuboidal shape, lamellar bodies and surfactant protein expression compared to submerged culture (Alcorn et al., 1997; Dobbs et al., 1997). It has further been shown that oxygen exposure promotes spontaneous differentiation of midgestation human fetal lung explant cultures in a dose-dependent manner (Acarregui et al., 1993). In the light of these findings, it can be hypothesized that ALI culture promoted the differentiation of iPSC-derived ATII-like cells by providing increased oxygen availability and facilitating epithelial cell polarization. Importantly, SFTPC+ cells were located on the apical surface of the airlifted cultures, consistent with a positive influence of air exposure on ATII-like differentiation. Interestingly, ABCA3+ cells, which are not detectable in human fetal lungs prior to 22-23 weeks of gestation, were present within our system (Stahlman et al., 2007). The cultures also expressed TM4SF1, which has been described as a marker for alveolar epithelial progenitor cells within the adult human lung (Zacharias et al., 2018). In addition, lamellar bodies, which are first detected during the saccular stage of human lung development, were present in the cultures (Baritussio et al., 1981; Chander et al., 1986; Van der Velden et al., 2013). Unlike in previous studies attempting to derive alveolar epithelial cells from hPSCs in 2D monoculture, cell detachment was not an issue in our ALI system (Gotoh et al., 2014; Huang et al., 2014). Taken together, these results confirm the feasibility of SFTPC+ ATII- like cell differentiation from iPSC-derived lung progenitor cells in ALI culture.

This work thereby shows, for the first time, a model that allows for the maturation of hPSC- derived ATII-like cells in an ALI culture format, providing a novel physiologically relevant platform for both basic research and drug discovery purposes. However, the in vitro system established in this thesis also faces certain limitations that will be discussed in the following paragraphs.

4.1.3. Cellular heterogeneity of the established iPSC-derived model system

The predominant limitation that presented itself in this work was the heterogeneity of iPSC- derived cultures and the inability to induce a pure population of alveolar epithelial cells via in vitro directed differentiation. In the future, in depth characterization, preferably by single- cell RNA-seq and immunohistochemistry should be performed to determine the exact identity of all non-ATII-like-cells in our model system to increase the interpretability of observations made utilizing these cultures. While the expression of airway markers like KRT5, MUC5AC and MUC5B in untreated cultures suggests that proximal airway epithelial cells were present in the model, immature progenitior cells of a fetal-like nature might also make up a certain proportion of the derived cells, as insufficient maturity is a commonly observed phenomenon in hPSC-derived systems (Baxter et al., 2015; Hrvatin et al., 2014; Koivumaki et al., 2018). In addition, another group recently found that mesenchymal cell populations of unknown origin were present in their hPSC-derived lung epithelial organoids (Chen et al., 2017). Hence, future characterization attempts should also include mesenchymal markers.

69 Discussion

Various other studies investigating the differentiation of ATII-like cells from hPSCs in 2D submerged culture or 3D organoids, have reported low induction efficiencies of SFTPC+ cells, even after applying sorting strategies to enrich for NKX2.1+ lung epithelial progenitors, or did not include any quantification of SFTPC+ cells in their reports (Chen et al., 2017; de Carvalho et al., 2019; Gotoh et al., 2014; Huang et al., 2014). This is in line with our observation, that only ~4% of the surface area of our iPSC-derived ALI cultures were positive for SFTPC and suggests that current directed differentiation regimes require further optimization in order to recapitulate human distal lung development more fully.

A more detailed understanding of the mechanisms underlying alveolar development in the human lung will likely result in refinement of in vitro differentiation strategies in the future. For instance, it was recently reported that a medium containing KGF, CHIR-99021, and RA supported the growth and maintenance of human primary fetal epithelial lung bud tip progenitors in vitro and likewise was capable of promoting an epithelial lung tip progenitor- like population derived from hPSCs. These hPSC-derived cells could be further enriched by needle-passaging and differentiation in the presence of KGF resulted in lung bud tip organoids containing 45% pro-SFTPC+ cells (Miller et al., 2018). Future efforts should therefore aim to optimize the growth factor conditions differentiating cells are exposed to in order to mimic human alveolar development more closely.

Alternatively, reporter lines could be utilized to achieve a higher percentage of ATII-like cells in our model system. Two recent studies have achieved a high percentage of SFTPC+ cells of ~50-70% in 3D culture by using SFTPC-reporter lines to purify ATII-like cells via FACS sorting (Jacob et al., 2017; Yamamoto et al., 2017). However, one group noted that SFTPC expression in alveolar organoids decreased over time and the cultures required repeated sorting of SFTPC+ cells followed by re-seeding every two weeks (Yamamoto et al., 2017).

Moreover, the identification of ATII-specific surface markers and the generation of commercially available antibodies against these proteins could enable a purification of the desired alveolar cells. Very recently, NaPi2b was identified as a candidate surface marker that allows for the enrichment of hPSC-derived SFTPC+ cells via FACS (Korogi et al., 2019). Interestingly, the authors also found that HTII-280, a surface marker commonly used to isolate ATII cells from primary adult lung tissue, was not applicable to the isolation of ATII cells from their hPSC-derived lung organoid system, as the number of HTII-280+ cells was too small for subculture or analysis (Gonzalez et al., 2010; Korogi et al., 2019). Therefore, it could be useful to apply NaPi2bhigh sorting to our model in the future to achieve a higher purity of iPSC-derived ATII-like cells without the requirement to establish reporter lines.

4.1.4. The apparent absence of ATI-like cells in iPSC-derived ALI cultures

A further drawback of the described culture system was the apparent absence of ATI-like cells. While the presence of a very small population of ATI-like cells in the iPSC-derived cultures could not be fully excluded, the assessment of bulk mRNA expression revealed no induction of CAV1, PDPN or AGER expression compared to iPSCs, suggesting the absence of ATI lineage differentiation. Despite the primary aim of this thesis to derive ATII cells, the lack of ATI cells limits the physiological relevance of our ALI cultures as a model of the alveolar epithelium. It further contradicts two previous reports utilizing similar differentiation regimes in which the presence of PDPN+ and AQP5+ cells was observed in hPSC-derived cultures (Huang et al., 2015; Yamamoto et al., 2017). While Aqp5 has been described as a protein specifically detected in differentiating ATI cells in murine systems, it was decided

70 Discussion not to utilize AQP5 as a marker of ATI cells in this work for two main reasons (Desai et al., 2014). Firstly, it has recently been shown that AQP5 expression is not specific to ATI cells during human lung development, being ubiquitously expressed in the low columnar epithelium that lines the developing alveolar ducts in the canalicular stage, and that AQP5 is not exclusively expressed by ATI cells in the adult human lung, but also by non-ciliated cells in the lower airways (Nikolic et al., 2017). Secondly, high expression levels of AQP5 were observed in undifferentiated iPSCs (data not shown), further limiting its suitability as a putative marker of differentiated epithelial lineages.

The absence of ATI marker expression despite the presence of ATII-like cells in our culture system was unexpected as mature ATII and ATI cells have been hypothesized to arise from the same bipotent progenitor during human lung development (Nikolic et al., 2017). This suggests a certain stimulus required for ATI differentiation could potentially be missing in our ALI system. Based on the literature, it could be assumed that the lack of mechanical stimuli that a developing mammalian lung is usually exposed to as a result of in utero breathing activity might be at least partly responsible for the lack of ATI cells in our static cell culture system (Rigatto, 1992). In line with this, Li at al. recently conducted in utero live imaging in mice and found the mechanical forces generated from the inhalation of amniotic fluid by fetal breathing movements to be essential for ATI cell differentiation (Li et al., 2018). In addition to its static nature, our in vitro system also fails to recapitulate the exact structural features of the alveolar compartment and is devoid of inter-cellular crosstalk with other cell types (e.g. endothelial cells, mesenchymal cells).

Interestingly, a related study aiming to derive alveolar epithelial cells from hPSCs faced similar issues in the respect that they found no evidence for the presence of PDPN+ or AGER+ ATI cells in their cultures, while ATII cell markers were abundantly expressed (Jacob et al., 2017). Primary ATII cells tend to rapidly lose their phenotype and transdifferentiate into ATI-like cells when cultured in vitro on tissue culture plastic in serum-containing medium (Bove et al., 2010; Dobbs, 1990; Fuchs et al., 2003). Based on this knowledge, Jacob et al. dissociated their hPSC-derived ATII organoids and re-seeded them under these conditions which resulted in a rapid decrease in SFTPC expression, accompanied by an upregulation of the ATI-associated transcripts CAV1, PDPN and AGER (Jacob et al., 2017). Likewise, an analogous experiment utilizing our ALI system led to a downregulation in ATII marker expression and an induction of CAV1, PDPN and AGER, confirming the ATI-like differentiation potential of the iPSC-derived cells. The fact that ATI-like differentiation was observed after multilayered ALI cultures were re-seeded onto a stiff plastic surface further substantiates the assumption that certain mechanical stimuli might indeed be required to aid the differentiation of hPSCs towards ATI cells.

4.2. Modeling aspects of IPF-related alveolar epithelial dysfunction in vitro utilizing iPSC-derived ALI cultures

4.2.1. Recapitulating IPF-related processes utilizing iPSC-derived ALI cultures

Following the optimization and characterization of the iPSC-derived ALI culture system, the effects of a pro-fibrotic cytokine environment on alveolar epithelial progenitors were investigated.

The disease specific milieu within the lung has been suggested to play a central role in the pathogenesis of IPF (Agostini and Gurrieri, 2006; Xu et al., 2016a). While there is

71 Discussion substantial evidence for the relevance of various cytokines and growth factors in IPF, including TGF-β, TGF-α, IL-13, TNF-α and IL-1β, the overexpression of which induces pulmonary fibrosis in animal models, no single factor is known to simultaneously activate all IPF-related pathways (B Moore et al., 2013; King et al., 2011). Despite this fact, most currently available in vitro models of pulmonary fibrosis rely on a single stimulus (Sundarakrishnan et al., 2018). To mimic a pro-fibrotic milieu in vitro, a novel cocktail (IPF- RC) containing nine IPF-relevant cytokines was designed and its effect on differentiating iPSC-derived distal lung epithelial progenitor cells was assessed (Barlo et al., 2011; Bellanger et al., 2016; Guiot et al., 2017; Lee et al., 2017; Meloni et al., 2004; Park et al., 2009; Tasaka et al., 2012; Vasakova et al., 2009; Zhang et al., 2016). As a primary aim of this work was to investigate the effect of a fibrosis-relevant environment on progenitors of the alveolar epithelium, IPF-RC stimulation was initiated on day 35 of the protocol, following induction of SFTPC expression, which constitutes a specific feature of alveolar epithelial progenitors in fetal human lungs and first becomes detectable after 15-17 weeks of gestation (Khoor et al., 1994; Nikolic et al., 2017; Treutlein et al., 2014).

The matrix metalloproteinases MMP-7 (a clinically used biomarker of IPF disease progression) and MMP-10 are elevated in IPF patients BAL and serum compared to healthy controls (Maher et al., 2017; Rosas et al., 2008; Sokai et al., 2015). Analysis of the culture medium in IPF-RC-treated cells showed increased MMP-7 and MMP-10 levels and RNA- seq analysis revealed an enrichment of transcripts involved in extracellular matrix organization, indicating an induction of IPF-relevant processes. Furthermore, a comparison of transcriptional changes in IPF-RC-treated iPSC-derived cultures and human IPF patient lungs revealed a significant overlap of up- and down-regulated transcripts (Kusko et al., 2016). On the other hand, there was also a substantial amount of non-overlapping deregulated genes, likely both due to the heterogeneity of our culture system and due to the fact that the publicly available IPF patients datasets were acquired from whole human lung homogenates. The analysis of whole lung homogenates contains multiple cell types (e.g. mesenchymal, endothelial and immune cells) that were not represented in our in vitro model system. This is supported by the presence of known epithelial-specific transcripts among the commonly deregulated genes, many of which have been associated with lung fibrosis. In accordance with previous reports, KLF4, VEGFA, IL32 and SLC19A3 were among the commonly downregulated transcripts (Hong et al., 2018; Lin et al., 2017; Murray et al., 2017; Xu et al., 2016b). BPIFB1 which was described to localize to the bronchiolized epithelium in the honeycomb cysts in usual interstitial pneumonia and MMP10, were among the commonly up-regulated transcripts (Bingle et al., 2013; Yang et al., 2013). The expression levels of CDH1, ACTA2 and COL1A1 remained unchanged, as assessed by qRT-PCR, providing no evidence for EMT taking place in our system. Taken together, our iPSC-derived in vitro model mimicked certain IPF-related changes in epithelial transcription and secretory phenotype.

In addition, RNA-seq data revealed many known airway associated transcripts with known roles in the developing and adult proximal lung, were among the top upregulated transcripts in IPF-RC-treated iPSC-derived cultures, e.g. SOX2, PAX9, TP63, KRT5, FOXJ1, SCGB1A1 and MUC5B, which have recently been confirmed as markers for the altered IPF lung epithelium via single-cell RNA-seq (Buendia-Roldan et al., 2016; McDonough et al., 2019; Plantier et al., 2011; Smirnova et al., 2016; Xu et al., 2016b; Yang et al., 2013). Upregulation of airway marker expression following IPF-RC stimulation was accompanied by a loss of ATII specific SFTPC expression and a shift in the expression of SOX2 and SOX9, two transcription factors known as central players in proximo-distal epithelial

72 Discussion patterning during human development (Nikolic et al., 2017; Nikolic et al., 2018). Mouse studies have revealed an essential role of Sox9 during branching morphogenesis, correct distal lung epithelial differentiation during development, as well as recovery of lung function after acute lung injury in adult mice (Li et al., 2015; Rockich et al., 2013). Expression of SOX2, which is associated with proximal airway rather than alveolar identity in healthy lungs, has been shown in the bronchiolized and enlarged distal airspaces in IPF (Plantier et al., 2011). Moreover, two murine studies have independently shown that selective overexpression of Sox2 in adult ATII cells resulted in an induction of conducting airway- related transcript expression in the alveoli (Kapere Ochieng et al., 2014; Tompkins et al., 2011). Immunohistological analysis of iPSC-derived cultures differentiated in the presence of IPF-RC revealed a significant reduction in SFTPC+ cell area and the emergence MUC5B+ secretory-like cells, an important role of which has been suggested in IPF. Not only has it been shown that MUC5B+ cells abundantly reside within the bronchiolized IPF epithelium, but a MUC5B promoter polymorphism, known to enhance MUC5B expression in the bronchiolo-alveolar epithelium, constitutes the strongest currently known genetic risk factor for IPF (Evans et al., 2016; Plantier et al., 2011; Seibold et al., 2013; Seibold et al., 2011). It was further demonstrated in this study, that highly aberrant direct transition zones between SFTPC+ and MUC5B+ epithelium exist within distal IPF patient lungs. Based on our findings in a human iPSC-derived alveolar epithelial model and IPF patient lungs, it can be hypothesized that alveolar epithelial progenitor cells could contribute to epithelial bronchiolization of the distal compartments of the IPF lung via aberrant trans-differentiation towards airway-like lineages. In the future, further investigation into this matter should be conducted using lineage tracing.

4.2.2. The role of individual cytokines in the induction of an IPF-related epithelial phenotype

In our model system, removal of IL-13, but not of TGF-β1 or TNF-α, from IPF-RC impaired its ability to promote differentiation towards airway-like cell fates at the cost of ATII-like cell differentiation, indicating IL-13 as one of the main drivers of the proximalization effect observed following IPF-RC stimulation. IL-13 has been described as a main mediator in respiratory diseases, particularly in asthma, where it is linked to mucus secretion and fibrogenic processes (Passalacqua et al., 2017). Moreover, IL-13 overexpression leads to pulmonary fibrosis in mice and it has been hypothesized that IL-13 antagonism could constitute a potential treatment strategy for IPF (Lee et al., 2001). However, while stimulation with IL-13 alone was sufficient to repress SFTPC expression in our study and induced FOXJ1 and TP63 expression, it failed to broadly recapitulate the influence of the IPF-RC on other airway-related or IPF-relevant transcripts. This is in line with the previous finding that IL-13 stimulation reduces SFTPC expression in primary human ATII cells (Seibold, 2018). IL-13 is also known to lead to cellular remodeling in the airway epithelium by inhibiting FOXJ1 and ciliated cell differentiation, opposed to its promoting effect on FOXJ1 expression in our model system, suggesting that IL-13 plays diverse roles in different lung epithelial cell types, which require further investigation (Ito and Mason, 2010). Our results are in accordance with the current consensus that neither IL-13, nor any other single factor is sufficient to simultaneously activate all IPF-related pathways in vitro and highlight the importance of more physiological human in vitro model systems (King et al., 2011). This is underlined by the recent failure of the phase 2 clinical study of tralokinumab, a human anti-IL-13 monoclonal antibody, in subjects with IPF (Parker et al., 2018).

73 Discussion

4.2.3. Small airway basal cells as a potential source of the bronchiolized epithelium in IPF

It has been proposed that migrating airway stem cells, rather than resident alveolar stem cells, are the stem cell source of the bronchiolized epithelium in IPF. As our model system contained rare KRT5+ basal-like cells in its untreated state, it cannot be excluded that the IPF-RC mediated airway-like phenotype of the cultures was in part a result of the expansion and differentiation of basal-like cells. To address this issue, primary SAECs were treated with IPF-RC during differentiation. In direct contrast to the iPSC-derived cultures, expression of SCGB1A1 and FOXJ1, both of which are upregulated in IPF lungs, were significantly repressed in IPF-RC-treated SAECs (Buendia-Roldan et al., 2016; Yang et al., 2013). In line with that, histological analysis and cilia beat measurements revealed a reduction in ciliation of SAEC cultures upon IPF-RC stimulation. Moreover, IPF-RC-induced a strong upregulation of MUC5AC, but not MUC5B in SAECs, contrary to the abundant emergence of aberrant MUC5B+ cells in the bronchiolized distal airspaces in IPF patients (Plantier et al., 2011; Seibold et al., 2013). These findings in primary basal cells provide indirect evidence that the airway-like phenotype observed in iPSC-derived IPF-RC cultures is unlikely the sole result of basal-like cell expansion and differentiation.

4.2.4. Limitations concerning the relevance of the established iPSC-derived model system of IPF-related alveolar epithelial dysfunction

In summary, the above-mentioned findings highlight that a synthetic IPF-related cytokine milieu has the potential to induce IPF biomarker secretion and skew alveolar epithelial differentiation towards airway cell phenotypes, demonstrating the capability of our iPSC- derived ALI model system to recapitulate certain aspects of alveolar epithelial dysfunction related to IPF. However, the described in vitro model system also has certain drawbacks that limit its relevance to the pathophysiological processes taking place in IPF patients.

The first limitation is attributed to the fact that the definite composition of the IPF BAL/ lung milieu is not known and even the combination of nine cytokines cannot fully mimic the environment epithelial cells are exposed to in a diseased lung. Moreover, while the described model could recapitulate certain aspects of the situation in late-stage disease by applying a mixture of cytokines that have been measured in patients’ lungs suffering from advanced IPF, this approach can only deliver limited information about the potential events taking place during disease onset. However, the usual interstitial pneumonia pattern in IPF is characterized by high temporal and spatial heterogeneity, meaning that some areas of the lung are very severely affected, while other areas are still maintained in a relatively healthy state (Smith et al., 2013). Therefore, our in vitro system utilizing an IPF-relevant cytokine cocktail, could hypothetically model the progression of epithelial dysfunction from severely affected towards healthy neighboring regions of the lung. This process might possibly occur via a distribution of the pro-fibrotic milieu produced in diseased fibrotic areas within the lung and exposure of epithelial progenitor cells in previously unaffected regions to this milieu. This aspect could hold critical relevance to the development of future therapeutic strategies, as to date, the majority of IPF patients do not experience an appropriate and timely diagnosis, due to a delay in seeking medical attention and a high frequency of misdiagnosis (Cosgrove et al., 2018). Halting disease progression towards the remaining functional areas of gas exchange in the advanced disease would constitute a clear benefit for patients, particularly in cases of strongly delayed diagnosis, by preventing further lung function decline.

74 Discussion

An additional drawback of the model utilized in this study lies in the lack of other disease relevant lung cell types besides epithelial cells. Our system fails to take into account the complex cellular interplay that leads to the vicious cycle of wound repair and scar formation that occurs in IPF. Most prominently, epithelial/fibroblast crosstalk is known to play central roles in IPF onset as well as disease progression, via the secretion of inflammatory factors and other paracrine signaling mechanisms (Selman and Pardo, 2002). In addition, our model leaves the role of mechanical tension which controls alveolar regeneration in vivo and is massively altered in IPF by the deposition of stiff extracellular matrix by myofibroblasts unconsidered (Chambers and Mercer, 2015; Liu et al., 2016). To take the aforementioned factors into account, the described iPSC-derived model could be expanded towards a co-culture system in the future. For example, primary healthy and IPF patient lung fibroblasts could be added into the basolateral compartment of the Transwell plate. This would also allow for an incorporation of differential disease-specific patient biology into the model. An alternative possibility could lie in a combination of iPSC-derived lung epithelium with iPSC-derived fibroblasts, the usefulness of which has recently been demonstrated in a study utilizing iPSC-derived mesenchymal organoids to model aspects of lung fibrosis (Wilkinson et al., 2017). As intercellular epithelial-mesenchymal crosstalk as well as interactions between epithelial cells and blood vessels have also been shown to play an important role during lung development, co-culture with other cell types (e.g. fetal human lung fibroblasts) during differentiation might also exert a positive influence on the maturation of iPSC-derived alveolar lineages (Volckaert and De Langhe, 2015; Yang et al., 2016).

An additional aspect that was not taken into account in the present study is the role of genetic risk factors in IPF pathogenesis. Apart from the most prominent genetic variant in the promotor region of MUC5B, other mutations, involving genes associated with surfactant production and telomere biology, have been associated with the disease (Winters et al., 2019). Thanks to the emergence of genome editing technologies like CRISPR/Cas9, it is now practical to introduce specific genetic alterations into pluripotent stem cells and future studies could thereby aim to combine our model system with an IPF-associated genetic background (Ding et al., 2013). The principle feasibility of this approach has recently been demonstrated in two studies that could partially recapitulate a specific autosomal recessive familiar form of pulmonary fibrosis in vitro utilizing genetically engineered hPSC-derived lung organoids (Chen et al., 2017; Korogi et al., 2019).

4.3. Conclusions and perspectives

This thesis shows for the first time the maturation of iPSC-derived lung epithelial progenitor cells towards alveolar epithelial cells in a 2D ALI culture format without the requirements of a surrounding 3D matrix or the adjacency of primary feeder cells. This provides a novel platform for both basic research and exploratory drug discovery purposes that allows for physiological air-exposure and good accessibility to the apical surface of the cultures. However, further efforts will need to be made to achieve a higher purity of ATII-like cells. Future studies uncovering the mechanisms underlying alveolar development in the human lung and the identification of robust ATII-specific surface markers will allow for a targeted refinement of culture conditions to increase the efficiency of iPSC-derived ATII cell generation. Until future insights enable refinements of the differentiation protocol and the achievement of a higher purity of ATII-like cells, in depth characterization of the described iPSC-derived model system by immunohistochemistry and single-cell RNA-seq should be

75 Discussion performed to increase the interpretability of observations made utilizing these cultures by revealing their exact cellular composition. In addition to increasing the yield of ATII-like cells, future efforts should aim at characterizing the derived cells further using functional assays, particularly to provide evidence for the production and secretion of mature surfactant proteins. Due to the inability of current hPSC-derived model systems to fully depict the phenotype and function of the adult human alveolar epithelium, all main findings made utilizing such systems, including those presented in this thesis, should ideally be confirmed in primary human ATII cells. Unfortunately, the practicability of such experiments and their inclusion into this thesis was not feasible due to limited access to human lung material.

Besides the cellular characteristics of the described model system, the composition of the culture medium, despite being serum-free, represents an additional limitation on the applicability of the iPSC-derived cultures. This is of particular relevance for pharmacological drug testing applications, as it contains a multitude of growth factors and small molecules that could potentially interact with drug candidates and their target pathways. For instance, the presence of 8-Br-cAMP, dexamethasone and the non-selective phosphodiesterase inhibitor IBMX in the terminal differentiation medium limits the suitability of the model as a testing platform for potentially anti-fibrotic compounds targeting cAMP synthesis or degradation, including the long-acting β2-adrenoceptor agonist olodaterol or the phosphodiesterase 4 inhibitor roflumilast (Cortijo et al., 2009; Herrmann et al., 2017). Future efforts should therefore be made to determine if the phenotype of iPSC-derived alveolar epithelial cultures can be maintained in the absence of pharmacologically active additives after the differentiation phase. If maintenance of the differentiated cells under these conditions were possible, this would create a sufficient time-window for compound testing in a more basic medium and increase the value of the system for drug discovery purposes.

Even though the established model system faces certain challenges, this thesis provides an important first proof-of-concept for the practical usefulness of iPSC-derived ALI cultures for modeling complex pathophysiological processes within the human lung. This was achieved by demonstrating that an IPF-relevant cytokine environment can skew iPSC- derived alveolar epithelial differentiation towards proximal lineages and induce disease- related transcriptional changes as well as secretion of IPF biomarkers. These findings suggest that aberrant trans-differentiation of epithelial stem cells in the fibrotic lung could disrupt the regular proximo-distal patterning of the lung epithelium and thereby contribute to the emergence of aberrant epithelial cell types, as well as the apparent bronchiolization in the distal IPF lung. However, in order to draw a definite conclusion about the potential role of alveolar-to-airway epithelial transdifferentiation the fate of individual pure alveolar epithelial progenitor cells will need to be traced to confirm the possibility of their transition into airway epithelial cells in the presence of pro-fibrotic stimuli. Further refinements of the described system, for instance by the incorporation of a FACS step to purify certain cell populations, might enable an evaluation of cell type-specific responses. Besides defining the exact cellular origin of observed changes in differentiation phenotypes, the identification of specific pathways that mediate this process would be of great interest.

As discussed above, an IPF-RC made up of various cytokines was utilized to mimic a pro- fibrotic environment in vitro as closely as possible, as no single factor is sufficient to simultaneously activate all IPF-related pathways according to the current consensus in the field. However, even a mixture of different recombinant cytokines is likely insufficient to fully capture the complexity and heterogeneity of altered alveolar cell biology and tissue microenvironment in IPF. In order to definitely relate the observations made in this thesis to

76 Discussion

IPF, the utilization of patient-derived BAL samples would represent the gold standard for a disease-relevant stimulus. However, the performance of such experiments in our laboratory is not feasible at present due to limited access to clinical material.

Despite its limitations, this thesis highlights the usefulness of complex human hPSC-derived in vitro culture systems as an alternative to primary human cells to model pathophysiological processes. This work further suggests that alveolar epithelial transdifferentiation may potentially play an important role in IPF disease progression. Based on the inability of nintedanib to reverse the bronchiolization-like phenotype in our iPSC-derived in vitro model, it may be hypothesized that this aspect might not be covered by currently available therapeutic strategies. This raises the need for further investigations to determine the exact pathophysiological mechanisms underlying alveolar epithelial cell dysfunction in IPF, which may open routes to novel therapeutic concepts not yet covered by current standard of care therapies.

77 Summary

5. SUMMARY

Limited access to primary human alveolar epithelial cells and the technical challenges associated with the in vitro maintenance and expansion of alveolar epithelial type II (ATII) cells, the proposed stem cells of the alveolar epithelium of the lung, have hindered advances in the understanding of the role of alveolar epithelial cells in lung homeostasis and disease. In recent years, directed differentiation of induced pluripotent stem cells (iPSCs) has emerged as a promising tool that allows for the de novo generation of specific human cell types from various different tissues, including alveolar epithelial cells of the lung.

In the first part of this work, directed differentiation of human iPSCs was applied to establish a novel cell culture model of ATII cell differentiation at air-liquid interface (ALI). Initially, NKX2.1+ lung epithelial progenitor cells were generated from healthy donor iPSCs. Temporal modulation of Wnt signaling was then utilized to promote distal lung differentiation. Importantly, lung progenitor maturation towards ATII-like cells was carried out on Transwell inserts and the apical culture surface was exposed to air to mimic the physiological environment of the alveolar epithelium. The ATII-like phenotype of the cells was confirmed by the expression of ATII marker proteins, including Surfactant protein C (SFTPC), and the presence of lamellar body-like structures. The cell culture system reported herein represents the first iPSC-derived model of the alveolar epithelium that encompasses long- term ALI culture.

In the second part of this study, the established differentiation protocol was utilized to develop a disease-relevant model system of epithelial dysfunction in idiopathic pulmonary fibrosis (IPF). IPF is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway-like epithelial cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems and the inability of existing animal models to sufficiently recapitulate IPF-related epithelial alterations. Recent reports have indicated that the pro-fibrotic cytokine milieu within the lung plays a critical role in the pathogenesis of IPF. Therefore, the iPSC-derived model system was stimulated with an IPF-relevant cytokine cocktail (IPF-RC) to investigate the effects of a pro-fibrotic environment on human alveolar epithelial progenitor cell differentiation. Stimulation with IPF-RC during differentiation increased secretion of IPF biomarkers and RNA-seq analysis of these cultures revealed significant overlap with human IPF patient data. IPF-RC treatment further impaired ATII differentiation by driving a shift towards an airway-like epithelial expression signature. These findings indicate that a pro-fibrotic cytokine environment has the potential to influence the proximo-distal differentiation pattern of human lung epithelial cells, suggestive of the pro-fibrotic milieu as a potential driver of alveolar bronchiolization in IPF. This study thereby describes, for the first time, the establishment of an iPSC-derived model system that recapitulates aspects of IPF-associated dysfunction of the human lung epithelium in vitro.

In conclusion, this thesis has led to the development of a novel disease relevant platform for both basic research and preclinical drug discovery applications that allows researchers to model aspects of epithelial dysfunction within the human lung. This study further demonstrates the high utility value of human iPSC-derived systems to study complex pathophysiological processes. The insights gained from this work are expected to have an impact on future investigations of epithelial dysfunction in chronic lung diseases and may contribute to the opening of routes to novel therapeutic concepts.

78 References

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95 Appendix

APPENDIX

Appendix A. PluriTest

Pluripotency testing of iPSC lines SFC065-03-03 and SFC084-03-01 was performed at Thermo Fisher Scientific using the Life Technologies Corporation PluriTest Service.

RNA purification: PureLink RNA Mini Kit (Cat. no. 12183025) DNAse Treatment: DNA-free Kit (Cat. no. AM1906) GeneChip Preparation: 250 ng total RNA

PluriTest Result: SFC065-03-03 and SFC084-03-01 were found to be pluripotent.

Pluripotency Plot:

The transcriptomes were analyzed and processed in the PluriTest algorithm to generate a pluripotency and novelty score. The red and blue background hint to the empirical distribution of the pluripotent (red) and non-pluripotent (blue) samples in the reference data set. An iPSC sample served as a positive control and a non-iPSC sample was included to serve as a non-pluripotent negative control. Pass shows clear pluripotency signature. Fail means the samples are not pluripotent.

Appendix B. Karyotyping

Molecular karyotyping of iPSC lines SFC065-03-03, SFC084-03-01 and SFC086-03-01 was carried out at LIFE & BRAIN GmbH, Bonn, Germany.

Technology used: Illumina BeadArray Product: HumanOmniExpressExome-8 BeadChip v1.3

Typing Scanner: Illumina iScan, S/N: N263

Genotype Analysis Genome Studio: GenomeStudio V2.0 Genotyping module: Ver. 2.0.0

96 Appendix iPSC line SFC065-03-03

Gender Chr. X derived: Male

Noteworthy findings: No larger chromosomal aberrations to be reported.

Karyogram:

97 Appendix iPSC line SFC084-03-01

Gender Chr. X derived: Female

Noteworthy findings: No larger chromosomal aberrations to be reported.

Karyogram:

98 Appendix iPSC line SFC086-03-01

Gender Chr. X derived: Female

Noteworthy findings: No larger chromosomal aberrations to be reported.

Karyogram:

99 Acknowledgements

ACKNOWLEDGEMENTS

Die Danksagung wurde aus Gründen des Datenschutzes in der veröffentlichten Dissertation entfernt.

100 Curriculum Vitae

CURRICULUM VITAE

Der Lebenslauf wurde aus Gründen des Datenschutzes in der veröffentlichten Dissertation entfernt.

101 Curriculum Vitae

Der Lebenslauf wurde aus Gründen des Datenschutzes in der veröffentlichten Dissertation entfernt.

102 Curriculum Vitae

Der Lebenslauf wurde aus Gründen des Datenschutzes in der veröffentlichten Dissertation entfernt.

103