Developing a 3D Model of the Airways

A thesis submitted for the degree of Doctor of Philosophy (PhD)

Tankut G. Güney

Airway Disease Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London SW3 6LY

Abstract

Chronic cigarette smoke exposure leads to chronic obstructive pulmonary disease (COPD) in susceptible individuals where goblet cell metaplasia, ciliated cell hypoplasia and extra cellular matrix (ECM) thickening are observed. Current in vitro COPD models such as air-liquid interface (ALI) cultures, cannot model the complex morphology and the cell-ECM interaction seen in vivo. No organoid models of COPD currently exist and, therefore, a lung organoid model termed bronchosphere of COPD was generated. Basal epithelial cells from normal healthy and COPD donors were cultured into bronchospheres over 20 days in 25% Matrigel. Bronchospheres were characterised using quantitative PCR, immunofluorescence and RNA sequencing. The effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function were investigated through a triple culture of bronchial epithelial, lung fibroblast and airway smooth muscle cells. COPD bronchospheres developed more slowly displaying goblet cell hyperplasia and ciliated cell hypoplasia with reduced cilial beat frequencies compared to normal healthy controls. Normal healthy basal cells chronically treated with cigarette smoke condensate formed bronchospheres with lumens lacking a differentiated epithelium. RNA-seq analysis of bronchospheres showed up-regulation of the club cell markers mucin 5B (muc5b) and secretoglobin family 3A member 1 (scgb3a1) in healthy vs COPD bronchospheres. Pathway analysis revealed increased extracellular matrix function and decreased fibroblast growth factor signalling in COPD and cigarette smoke-treated healthy bronchospheres, which may be a possible driver of disease phenotype. Epithelial-stromal cross talk enabled formation of epithelial cell-driven branching tubules consisting of luminal epithelial cells surrounded by stromal cells. Addition of agarose to the Matrigel scaffold (Agrigel) altered the matrix viscoelasticity and stiffness and prevented tubule collapse. This thesis described the development of a novel bronchosphere model of COPD derived from primary human airway cells that recapitulates many functions of human COPD. Generating large numbers of these bronchospheres provides opportunities for future personalised drug testing. 2

Declaration of originality I, Tankut G. Güney declare that I wrote this thesis and the experiments and work described herein, except where appropriately referenced, was performed by myself. Information derived from the published and unpublished work of others has been acknowledged in the text and full references are given.

Copyright declaration ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’.

Acknowledgements

I would like to give my sincerest heartfelt thank you to my supervisors Professor Ian Adcock and Dr Sharon Mumby for their tremendous support over the past 4 years. Thank you for sharing your knowledge and giving me the space to drive this project forward. I would like to thank my secondary supervisor Dr Mark Dowling and the NIBR team Dr Kevin White and Kaushik Subramanian for helping me formulate a project and making the best of my time in Boston - I had a blast! I would also like to thank Dr Vera Ruda for running the RNA-seq. I would like to extend my gratitude to Dr Iain Dunlop and Alfonso Herranz for helping me formulate a new scaffold and letting me use their facilities. My thanks also go to Vahid Elyasigomari for showing me how to do RNA-seq analysis. I would also like to thank Dr Rebecca Holloway for allowing me to teach the knowledge I gained in lab at Outreach. You really allowed me to develop a passion for teaching. Furthermore, I would like to thank my friends Dr Sarah Christen, Dr Elaina Maginn, Dr Abel Tesfai, Nicholas Wood and Dr Elisa Zanini for pulling me out of lab and showing me a world beyond my work. Thank you to Daniel Browne and Miranda Debenham for their enthusiasm in listening to updates on my project at our weekly ‘Lung News’ segment during our D&D games. Also, to Dr Nathalie Schmidt for her insight during our coffee meetings. Thank you to Christian and Dr Andrea Dungl for their support during my 4 years. Finally, words cannot express how immensely grateful I am to my fiancée Daniela Dungl and to my mother Betigül Güney who have shown love, patience, kindness and understanding during my PhD. A special thank you to my mother who has always championed me and pushed me to achieve my best. I dedicate this thesis to you both.

Table of Contents

Abbreviations ...... 15

Chapter 1 Introduction ...... 26

1.1 Chronic obstructive pulmonary disease (COPD) ...... 27

1.1.1 Definition, Risk factors and prevalence ...... 27

1.1.2 COPD Pathophysiology ...... 29

1.2 Lung Morphology and Development ...... 31

1.2.1 Overview of Lung Development ...... 31

1.2.2 Branching Morphogenesis ...... 32

1.2.3 The Extracellular Matrix (ECM) ...... 37

1.2.4 Luminogenesis ...... 45

1.3 Lung Epithelial Cell Populations ...... 50

1.3.1 Airway Smooth Muscle (ASM) Development ...... 53

1.4 Airway Regeneration, Repair and COPD...... 54

1.5 Current Models of the Airway ...... 58

1.5.1 Murine Models ...... 59

1.5.2 2D Models of Lung Disease ...... 59

1.5.3 Organoid Models ...... 61

1.5.4 Future Directions ...... 64

1.6 Hypothesis ...... 66

1.6.1 Aims ...... 67

2 Chapter 2 Methodology ...... 68

2.1 Monolayer Cell Culture ...... 69

2.1.1 HBE cell culture...... 69

2.1.2 Stromal cell culture ...... 70 5

2.1.3 Feeder Layer Culture ...... 71

2.1.4 HBE-3T3 cell co-culture ...... 71

2.2 Bronchosphere Culture ...... 73

2.3 Bronchotubule Culture ...... 74

2.3.1 Matrigel Overlay Culture ...... 74

2.3.2 Matrigel Triple Culture ...... 75

2.3.3 Agrigel* (agarose-matrigel mix) Culture ...... 76

2.4 Generation of Cigarette Smoke Condensate (CSC) ...... 78

2.5 Cell Viability Assay ...... 79

2.6 Cilia Beat Frequency (CBF) ...... 79

2.7 Bronchosphere RNA Extraction ...... 80

2.7.1 Bronchosphere Isolation, lysis and homogenisation ...... 80

2.7.2 RNA Extraction ...... 81

2.7.3 Reverse transcription ...... 82

2.8 Immunofluorescence (IF) Staining ...... 83

2.9 Maxiprep Plasmid Purification and Plasmid Generation ...... 84

2.9.1 Lentiviral Generation ...... 86

2.10Rheometry of Agarose, Matrigel and Agrigel ...... 88

2.11RNA-Seq Bioinformatics ...... 89

2.12Statistical Analysis ...... 89

3 Chapter 3 Bronchosphere Model Development and Characterisation ..... 90

3.0 Introduction ...... 91

3.1 Donor Characterisation ...... 93

3.2 Developing NHBE and CHBE Bronchosphere Model ...... 97

3.3 Developing a Chronic Smoking Bronchosphere Model ...... 108

3.4 Discussion ...... 118

4 Chapter 4 Global Expression of Transcripts in Bronchospheres ...... 121

4.0 Introduction ...... 122

4.1 Quality Control of Bronchosphere RNA ...... 123

4.2 Differences between COPD (CHBE-B) and normal healthy bronchospheres (NHBE-B) at day 0 ...... 123

4.3 Effect of Bronchosphere Culture on NHBE and CHBE bronchospheres at Day 8 ...... 130

4.4 Effect of Bronchosphere Culture on NHBE and CHBE bronchospheres at day 20 compared to day 0 ...... 143

4.5 Effect of cigarette smoke condensate (CSC) on gene expression profiles over time in normal healthy human bronchospheres (NHBE-B)...... 153

4.6 Discussion ...... 167

5 Chapter 5 Effect of Epithelial-Stromal Cell Interaction in Bronchosphere Culture ...... 173

5.1 Epithelial-Stromal Interaction in 3 Dimensions ...... 174

5.2 NHBE-NHLF co-culture results in tubular branching organoids ...... 176

5.3 NHBE-NHLF-NHASM Triple Culture ...... 180

5.4 Scaffold Optimisation ...... 182

5.5 NHLF and NHASM YFP and mCherry Transduction ...... 189

5.6 Agrigel Triple Culture ...... 190

6 Chapter 6 General Discussion ...... 197

6.0 General Discussion ...... 198

7 Chapter 7 References ...... 205

8 Chapter 8 Appendix ...... 234

8.0 Image User Licence and Permission ...... 235

List of Figures

Chapter 1 Figure 1.0.0: Emphasymatic destruction of alveoli ...... 31 Figure 1.2.1: Stages of lung development ...... 31 Figure 1.2.2: Tracheal tube morphogenesis from the anterior foregut of mouse embryo ...... 34 Figure 1.2.3: Epithelial-stromal cell signalling during branching morphogenesis ..... 36 Figure 1.2.4: Lung tubule extension ...... 37 Figure 1.2.5: Proteins in the extracellular matrix (ECM ...... 38 Figure 1.2.6: Basement membrane architecture self assembly ...... 39 Figure 1.2.7: Matrix metalloproteinases (MMPs ...... 31 Figure 1.2.8: Growth Factor and heparin sulphate (HS) interactions ...... 43 Figure 1.2.9: Structure of laminin ...... 45 Figure 1.2.10: Luminogenesis via vesicular trafficking in MCDK cysts (Hollowing) .. 48 Figure 1.2.11: Luminogenesis by cavitation ...... 50 Figure 1.3.1: Lung Epithelial population ...... 52 Figure 1.5.2.1: Schematic of the lung-on-a-chip model by Benam et. al ...... 62 Figure 1.5.2.2: Lung organoid ...... 64

Chapter 2 Figure 2.1: Schematic of bronchosphere culture ...... 75 Figure 2.2: Schematic of Matrigel overlay culture ...... 76 Figure 2.3: Schematic of Matrigel triple culture ...... 77 Figure 2.4: Schematic of Matrigel overlay culture ...... 78 Figure 2.9: Schematic of yellow fluorescence protein (YFP) and mCherry plasmids ...... 86

Chapter 3 Figure 3.1.1: Quantitative PCR of basal cell markers in donor cells ...... 95 Figure 3.1.2: Normal Human bronchial epithelial (NHBE) donor cell protein expression of basal cell markers ...... 96

Figure 3.1.3: COPD human bronchial epithelial (CHBE) donor cell protein expression of basal cell markers ...... 97 Figure 3.2.1: Effect of plastic on spheroid development ...... 98 Figure 3.2.2: Cell Density Assay ...... 99 Figure 3.2.3: Bronchosphere development, Bronchospheres/well and Lumen sizes of bronchospheres from normal human bronchial epithelial cells (NHBE-B) and from bronchial HBEs from COPD patients (CHBE-B) ...... 100 Figure 3.2.4: Development of normal healthy human bronchial epithelial (NHBE-B) and COPD human bronchial epithelial bronchospheres (CHBE-B) during 20 days of culture ...... 103 Figure 3.2.5: Immunofluorescence staining of human airways ...... 104 Figure 3.2.6: Development of normal healthy human bronchial epithelial (NHBE-B) and COPD human bronchial epithelial bronchospheres (CHBE-B) during 20 days of culture ...... 106 Figure 3.2.7: Gene expression of normal healthy human bronchial epithelial (NHBE-B) and COPD human bronchial epithelial bronchospheres (CHBE-B) during 20 days of culture ...... 108 Figure 3.3.1: Cigarette smoke condensate (CSC) treatment of normal healthy human bronchial epithelial cells (NHBE) over 8 days of culture ...... 110 Figure 3.3.2: Development of DMSO- or cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (DMSO-NHBE-B and CSC-NHBE-B respectively) during 20 days of culture ...... 113 Figure 3.3.3: Development of DMSO- or cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (DMSO-NHBE-B or CSC-NHBE-B respectively) during 20 days of culture ...... 116 Figure 3.3.4: Development of DMSO- or cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (DMSO-NHBE-B and CSC-NHBE-B respectively) during 20 days of culture ...... 118

Chapter 4 Figure 4.2.1: Baseline differential gene expression ...... 125 Figure 4.3.1: Differential gene expression after 8 Days of bronchosphere culture . 131 Figure 4.4.1: Differential gene expression after 20 Days of bronchosphere culture 144 9

Figure 4.5.1: Baseline differential gene expression ...... 154 Figure 4.5.2: Differential gene expression after 20 Days of bronchosphere culture ...... 160 Figure 4.6.1: Response of cells to REDOX switching...... 168

Chapter 5 Figure 5.0.0: Airway wall structure ...... 176 Figure 5.1.1: Normal healthy human bronchial epithelial (NHBE) cell and normal healthy human lung fibroblast (NHLF) co-culture ...... 178 Figure 5.1.2: Bronchotubule formation ...... 179 Figure 5.1.3: Bronchotubule morphology ...... 180 Figure 5.1.4: Cell behaviour above gel ...... 180 Figure 5.2.1: NHBE concentration optimisation ...... 181 Figure 5.2.2: Normal healthy human airway smooth muscle (HASM) Contraction assay ...... 182 Figure 5.3.1: Viscoelastic properties of agarose gel scaffold ...... 183 Figure 5.3.2: Viscoelastic properties of agrigel scaffold ...... 185 Figure 5.3.3: Comparison of viscoelastic characteristics of agarose and agrigel ... 187 Figure 5.3.4: Laminin staining of Agrigel ...... 188 Figure 5.3.5: Scaffold optimisation ...... 189 Figure 5.4.1: Stromal Cell Lentiviral Transduction ...... 190 Figure 5.5.1: Bronchotubule Triple Culture in 0.5/25% Agrigel ...... 192 Figure 5.5.2: Bronchotubule Triple Culture in 0.5/25% Agrigel Day 20 ...... 193

Chapter 6 Figure 6.1: Summary of Models ...... 205

List of Tables

Chapter 1 Table 1.0.0 ...... 29

Chapter 2 Table 2.1.1: Bronchial epithelium cell growth medium (BEGM) ...... 70 Table 2.1.2: Complete DMEM ...... 71 Table 2.1.3: 3T3 medium ...... 72 Table 2.1.4: Co-culture medium (CCM) ...... 72 Table 2.2: Differentiation medium ...... 74 Table 2.4.1: CSC reagents ...... 79 Table 2.5: MTT reagents ...... 80 Table 2.7.1: Isolation, lysis and homogenisation reagents ...... 81 Table 2.7.2: RNA extraction reagents ...... 82 Table 2.7.3: Reverse polymerase chain reaction components ...... 83 Table 2.8: Immunofluorescence reagents ...... 84 Table 2.9.1: Plasmid reagents ...... 85 Table 2.9.2: Lentiviral reagents ...... 87 Table 2.9.3: Components, concentrations and volume of viral titre real time PCR master mix ...... 89

Chapter 3 Table 3.1.1: Normal Human bronchial epithelial (NHBE) cell donor demographics . 94 Table 3.1.2: COPD Human bronchial epithelial (CHBE) cell donor demographics ... 94

Chapter 4 Table 4.2.1: Top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) and bronchospheres derived from normal healthy human epithelial (NHBE) cell (NHBE-B) ranked according to log2 fold-change at day 0 of culture ...... 127 Table 4.2.2: Top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) and bronchospheres 11

derived from normal healthy human epithelial (NHBE) cell (NHBE-B) ranked according to adjusted p-value change at day 0 of culture ...... 128 Table 4.2.3: Pathway analysis of all the genes increased or decreased by ≥2-fold in COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) and bronchospheres derived from normal healthy human epithelial (NHBE) cell (NHBE-B) at day 0 of culture ...... 130 Table 4.3.1: Top differentially expressed genes between normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 8 and NHBE at day 0 ranked according to log2 fold-change ...... 133 Table 4.3.2: Up and down regulated genes in normal human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 8 versus NHBE at day 0 of culture ...... 134 Table 4.3.3: Pathway analysis of all the genes increased or decreased by ≥2-fold in normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 8 compared to day 0 of culture ...... 136 Table 4.3.4: Top differentially expressed genes between COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) at day 8 versus day 0 ranked according to log2 fold-change ...... 138 Table 4.3.5: Up and down regulated genes in COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) at day 8 versus day 0 ...... 139 Table 4.3.6: Pathway analysis of all the genes increased or decreased by ≥2-fold in COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) at day 8 compared to day 0 ...... 141 Table 4.3.7: Top differentially expressed genes between COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) between day 8 and day 0 compared to the top differentially expressed genes in normal healthy human bronchial epithelial (NHBE) cell derived bronchospheres (NHBE-B) between day 8 and day 0 ranked according to log2 fold-change ...... 143 Table 4.4.1: Top differentially expressed genes between normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 20 compared to day 0 ranked according to log2 fold-change ...... 145

Table 4.4.2: Up and down regulated genes in normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 20 versus day 0 ...... 146 Table 4.4.3: Pathway analysis of all the genes increased or decreased by ≥2-fold in normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 20 compared to day 0 ...... 148 Table 4.4.4: Top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) at day 20 and day 0 ranked according to log2 fold-change ...... 150 Table 4.4.5: The top 15 Up and down regulated genes in COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) at day 20 versus day 0 ranked by adjusted p value ...... 151 Table 4.4.6: Pathway analysis of all the genes increased or decreased by ≥2-fold in COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) day 20 compared to day 0 ...... 153 Table 4.5.1: Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) and control NHBE-B at day 0 ranked according to log2 fold-change ...... 156 Table 4.5.2: Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) and control bronchospheres (NHBE-B) at day 0 ranked according to log2 fold-change ...... 157 Table 4.5.3: Pathway analysis of all the genes increased or decreased by ≥2-fold in cigarette smoke (CSC)-treated normal healthy human epithelial (NHBE)-derived bronchospheres (CSC-NHBE-B) at day 0 compared control NHBE-B at day 0 ...... 159 Table 4.5.4: Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) at day 20 and control bronchospheres NHBE-B at day 0 ranked according to log2 fold-change ...... 162 Table 4.5.5: Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial 13

(NHBE) cell-derived bronchospheres (CSC-NHBE-B) at day 20 and control bronchospheres (NHBE) at day 0 ranked according to log2 fold-change ...... 163 Table 4.5.6: Pathway analysis of all the genes increased or decreased by ≥2-fold in cigarette smoke (CSC)-treated normal healthy human epithelial (NHBE)-derived bronchospheres (CSC-NHBE-B) at day 20 compared control NHBE-B at day 0 ...... 165 Table 4.5.7: Comparison of top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B), normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) and cigarette smoke condensate (CSC)-treated NHBE-B (CSC-NHBE-B) ranked according to log2 fold-change at day 20 compared to day 0 ...... 167

Abbreviations

ADAM a Disintegrin and Metalloproteinase

ADH1C Alcohol Dehydrogenase 1C (class I), Gamma Polypeptide

AEC Airway Epithelial Cells

AFE Anterior Foregut Endoderm

ALDH1L2 Aldehyde Dehydrogenase 1 Family Member L2

ALI Air Liquid Interface Culture

AMIS Apical Membrane Initiation Site

ANKRD66 Ankyrin Repeat Domain 66

ANX (2 or A10) Annexin (2 or A10)

ALOX5 Arachidonate 5-Lipoxygenase

ALOX15 Arachidonate 15-Lipoxygenase

AKT AK Transforming

APH1 Anterior Pharynx Defective 1 aPKC Atypical Protein Kinase C

APOA1 Apolipoprotein A1

ASCL1 Achaete-Scute Homolog 1

ASM Airway Smooth Muscle

ASMC Airway Smooth Muscle Cells

ATI Alveolar Epithelial Type 1

ATII Alveolar Epithelial Type 2

ATPase Adenine Triphosphatase

ATOH8 Atonal bHLH Transcription Factor 8

BCL-2 B-cell Lymphoma 2

β-ME β-mercaptoethanol

BEGM Basal Epithelial Growth Media

BIF SH3-Domain GRB2 Like Endophilin B1

BM Basement Membrane

BIM BCL2-like protein 11

BMP Bone Morphogenetic Protein

BMP3 Bone Morphogenetic Protein 3

BMP4 Bone Morphogen Protein 4

BMPR BMP Receptor

BPIFA2 BPI Fold Containing Family A Member 2

BPIFB1 BPI Fold Containing Family B Member 1

C9orf135 Chromosome 9 Open Reading Frame 135

C20orf85 Chromosome 20 Open Reading Frame 85

CAPN13 Calpain 13

CEACAM6 Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6

CBF Cilial Beat Frequency

CCBE1 Collagen and Calcium Binding EGF Domains 1

CCM Co-Culture Medium

CCR Cell Recovery Solution

CDC42 Cell Division Cycle 42

CFAP74 Cilia and Flagella Associated Protein 74

CFAP221 Cilia and Flagella Associated Protein 221

CFP Cambridge FiGlter pad

CFTR Cystic Fibrosis Trans Membrane Conductance Regulator

CHAC1 ChaC Glutathione Specific Gamma- Glutamylcyclotransferase 1

CHBE COPD Human Bronchial Epithelial Cells

CHBE-B CHBE Bronchosphere

COPD Chronic Obstructive Pulmonary Disease

CGRP Calcitonin Gene Related Protein

CRB Crumbs

CS Cigarette Smoke

CSF2 Colony Stimulating Factor 2

CSC Cigarette Smoke Condensate

CTNNB1 Catenin B1

CXCL8 Interleukin 8

CYP2F1 Cytochrome P450 Family 2 Subfamily F Member 1

DKK1 Dickkopf WNT Signalling Pathway Inhibitor 1

DLL 1 Delta Like Ligand 1

DLL 3 Delta Like Ligand 3

DLL 4 Delta Like Ligand 4

DHRS9 Dehydrogenase/reductase 9

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethyl Sulfoxide

DRC7 Dynein Regulatory Complex Subunit 7

DUSP1 Dual Specificity Phosphatase 1

ECM Extra Cellular Matrix

EDTA Ethylenediaminetetraacetic Acid

EGF Epidermal Growth Factor

EMT Epithelial to Mesenchymal Transition

ELISA Enzyme Linked Immunosorbent Assay

EXOC 4 Exocyst Complex Component 4

EXOC 5 Exocyst Complex Component 5

FAM92B Family with Sequence Similarity 92 Member B

FeV1 Forced Expiratory Volume

FGF2 Fibroblast Growth Factor 2

FGF7 Fibroblast Growth Factor 7

FGF9 Fibroblast Growth Factor 9

FGF10 Fibroblast Growth Factor 10

FGFR1 FGF Receptor 1

FGFR2b FGF Receptor 2b

FGFR3 FGF Receptor 3

FGFR4 FGF Receptor 4

FMO2 Flavin Containing Monooxygenase 2

FMO6P Flavin Containing Monooxygenase 6 Pseudogene

FOSB FosB Proto-oncogene, AP-1 Transcription Factor Subunit

FOXOA2 Forkhead Box A2

FOXF1 Forkhead Box F1

FOXJ1 Forkhead Box j 1 18

FZD Frizzled Class Receptor

FSTL1 Follistalin Like 1

FVC Forced Vital Capacity

G’ Storage Modulus

G” Loss Modulus

GAG Glycosaminoglycan

GFR Growth Factor Reduced

GOLD Global Initiative for Chronic Obstructive Lung Disease

GSH Glutathione

GLB1L3 Galactosidase beta 1 like 3

GLI (1 or 3) Glioma Associated Homologue (1 or 3)

GMCSF Granulocyte Macrophage Colony Stimulating Factor

GSSG Glutathione Disulfide

GSTA1 Glutathione S-transferase Alpha 1

GSTA2 Glutathione S-transferase Alpha 2

GTSE1 G2 and S-phase expressed 1

GSVA Gene Set Variation Analysis

HDAC- (1 or 2) Histone Deacetylase (1 or 2)

HES (1 or 5) Hairy Enhancer of Split (1 or 5) hPSC Human Pluripotent STEM Cell

HS Heparan Sulphate

HSPG Heperan Sulphate Proteoglycan

HBE Human Bronchial Epithelial Cells

HBSS Hank’s Balanced Salt Solution 19

ID2 SOX9/Inhibitor of DNA Binding 2

IL1RL1 Interleukin 1 Receptor Like 1

IL1R2 Interleukin 1 Receptor Type 2

IL-6 Interleukin 6

IL-13 Interleukin 13

IL13RA2 Interleukin 13 Receptor Subunit Alpha 2

IL36B Interleukin 36 Beta

IPF Idiopathic Lung Fibrosis

ITGA6 Integrin Subunit α 6

JAG (1 or 2) Jagged (1 or 2)

JAK1 Janus Kinase 1

JAM Junctional Adhesion Molecule

KI67 Marker of Proliferation Ki-67

KIF(14, 18B or 20A) Kinesin Family Member (14, 18B or 20A)

KIT KIT Proto-oncogene Receptor Tyrosine Kinase

KRT5 Keratin 5

LG Laminin G Domain-Like

LB Lauria-Bertani Broth

LBO Lung Bud Organoid

LGN Lateral Geniculate Nucleus

LGR5 Leucine-rich Repeat-Containing G-protein Coupled Receptor 5

LBO Lung Bud Organoids

LOC Lung on a Chip

LOXL4 Lysyl Oxidase Like 4

L4 Laminin L4 terminal

LE (a, b or c) Laminin-type Epidermal Growth Factor Like (a, b, or c)

LF Laminin LF Domain

LG (1, 2, 3, 4 or 5) Laminin G Domain Like (1, 2, 3, 4 or 5)

LPS Lipopolysaccharide

LRRC10B Leucine Rich Repeat Containing 10B

MAML- (1, 2 or 3) Mastermind (1, 2 or 3)

MAPK Mitogen Activated Protein Kinase

MCC Mucociliary Clearance

MDCK Mardin-Darby Canine Kidney

MET Mesenchymal to Epithelial Transition

MMP(1, 2, 8, 9 or 12) Matrix Metallo Proteinase (1, 2, 8, 9, or 12)

MOI Multiplicity of Infection

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide

MS4A8 Membrane Spanning 4-Domains A8

MUC5AC Mucin 5 A/C

MUC5B Mucin 5 B

MUC4 Mucin 4

MUC20 Mucin 20

NCSTN Nicastrin

NE Neuroendocrine Cells

NECD Notch Extracellular Domain

NEXT Notch Extracellular Truncated Form

NGFR Nerve Growth Factor

NHASM Normal Human Airway Smooth Muscle

NHBE Normal Human Bronchial Epithelial Cells

NHBE-B NHBE Bronchosphere

NHLF Normal Human Lung Fibroblasts

NCID Notch Intracellular Domain

NLGN4Y Neuroligin 4 Y-linked

NKX2-1 NK2 Homeobox 1

N-MYC N-MYC Proto-Oncogene Protein

NRF2 Nuclear Factor Erythroid Derived 2 Like 2

NTN1 Netrin 1

NUMA Nuclear Mitotic Apparatus Protein 1

OD Optical Density

ODC1 Ornithine Decarboxylase 1

OSM Oncostatin M

P21 Cyclin Dependent Kinase 1A

P63 Tumour Protein 63

PALS1 Membrane Palmitoylated Protein 5

PAR 3 Partitioning Defective 3

PBS Phosphate Buffered Saline

PCR Polymerase chain reaction

PDGFR Platelet Derived Growth Factor Receptor

PAR Partitioning-defective 22

PBX1 PBX Homeobox 1

PEG Poly Ethylene Glycol

PEGDA Poly Ethylene Glycol Diacarlate

PGA Polyglycolide

PEN2 Presenilin Enhancer 2

PI3K Phosphatidylinositol-4,5-Bisphosphate 3-kinase

PIGR Polymeric Immunoglobulin Receptor

PIP2 Phosphatidylinositol-4,5-Bisphosphate

PTCHD Patched

PSEN1 Presenilin 1

POFUT1 Protein O-Fucosyl Transferase 1

PVDF Polyvinylidene Fluoride

QPCR Quantitative PCR

RA Retinoic Acid

RAC1 Ras-Related C3 Botulinum Toxin Substrate 1

RHO (A) Ras homolog Gene Family Member (A)

RPBJκ Recombination Signal Binding Protein for Immunoglobin Jκ

ROCK1 Rho-associated Protein Kinase 1

ROS Reactive Oxygen Species

RPS4Y1 Ribosomal Protein S4 Y-Linked 1

RSV Respiratory Syncytial Virus

SCGB3A1 Secretoglobin 3A 1

SCGB3A2 Secretoglobin 3A 2

SCRIB Scribble

SH3 SH3 (SRC Homology 3) domain binding protein 4

SHH Sonic Hedgehog

SLC34A2 Solute Carrier Family 34 Member 2

SNARE Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor

SO2 Sulfur Dioxide

SOX (2 or 9) SRY (Sex Determining Region Y)-Box (2 or 9)

SPAG6 Sperm Associated Antigen 6

ST6GAL1 ST6 Beta-galactoside Alpha-2,6-sialyltransferase 1

STAT (3) Signal Transducer and Activator of Transcription (3)

TAZ Tafazzin

TBX (4) T-Box (4)

TEER Transepithelial Electrical Resistance

TGF-β Transforming Growth Factor

TNF-α Tumour Necrosis Factor

TNS Trypsin Neutralising Solution

TPM Total Particulate Matter

Th2 T-helper Cell 2

UPP1 Uridine Phosphorylase 1

ULA Ultra Low Attachment

UTY Ubiquitously Transcribed Tetratricopeptide Repeat

WNT (2, 2a, 5a or 7b) Wingless-related MMTV (mouse mammary tumour virus) Integration Site (2, 2a, 5a or 7b)

XIST X Inactive Specific Transcript

YAP Yes Associated Protein

YFP Yellow Fluorescence Protein

Chapter 1

Introduction

1.1 Chronic obstructive pulmonary disease (COPD)

1.1.1 Definition, Risk factors and prevalence COPD is a complex, multifactorial and progressive syndrome of the lung characterised by persistent irreversible airflow limitation due to chronic inflammation, hyper mucous secretion and alveolar degradation from exposure of the airways to noxious gasses particularly cigarette smoke (Buist et al., 2007). The severity of the disease changes depending on exacerbations and/or comorbidities (Rabe et al., 2007).

COPD is the 3rd leading cause of morbidity and mortality globally affecting over 384 million people and killing over 3.2 million people every year (GOLD, Global Initiative for Chronic Obstructive Lung Disease, https://goldcopd.org/). The incidence of COPD globally has been increased due to the recent recognition of 100 million COPD subjects in China (Wang et al., 2018). The world health organisation estimates that COPD will be the third leading cause of death by 2030. The prevalence of COPD cannot be fully calculated as misdiagnosis is common due to the similarity of COPD symptoms with other lung conditions and the failure to use spirometry (Lozano et al., 2012, Burney et al., 2015).

Patients are considered for COPD diagnosis when presenting with dyspnoea, chronic cough, sputum production and/or history of exposure to risk factors such as smoking. Lung function (spirometry) is then essential to clinically diagnose COPD, where the ratio of forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) measurements (FEV1: FVC) is compared to a reference value based on age, height, sex, and race. Airflow limitation is present in COPD patients with decreased FEV1: FVC ratios <70% (Table 1). Due to the complex heterogenous nature of COPD, a single FEV1 test cannot be used as the only diagnostic parameter for COPD (Lange et al., 2015, Bolton et al., 2013). The recent GOLD Guidelines (https://goldcopd.org/) has changed the definition to place greater emphasis on symptoms and risk of exacerbations although lung function remains a critical determinant of COPD (Soriano JB, 2018).

The impact of symptoms on a patient’s quality of life (QoL) is assessed using the COPD assessment test (CAT) questionnaire. A score of 0-40 is given according 27

to answers where score of ≥10 indicates a high impact of symptoms on QoL (Table 1.0.0). Risk is defined by the patient’s exacerbation history where two or more exacerbations in the preceding year are considered as high risk (Vestbo et al., 2012).

6-minute walk tests are used to measure the functional impact of COPD on the patient’s health status and survival. The test is simple to perform since patients are asked to walk for 6 minutes up and down a marked 30m path and the distance covered measured (Bolton et al., 2013).

The requirement for multiple tests highlights the difficulty in disease diagnosis. To improve and simplify COPD diagnosis and treatment the use of phenotyping including the noting of associated comorbidities has been proposed. Phenotyping has been defined as:

‘a single combination of disease attributes that describe differences between individuals with COPD as they relate to clinically meaningful outcomes like symptoms, exacerbations, response to therapy, rate of disease progression or death.’(Han et al., 2010).

Table 1.0.0: COPD categories based on symptom, spirometry and number of exacerbations. Spirometry can be used to classify disease severity using FEV1%. However, must be used in conjunction with other tests such as CAT or mMRC which assess symptoms and exacerbation risk (Lange et al., 2015).

Stage FEV % mMRC Patient 1 FEV /FVC Severity Exacerbations/Year CAT (GOLD) Predicted 1 Score

A I 80≤FEV1 <0.70 Mild ≤1 <10 0-1

B II 80≤FEV1 <0.70 Moderate ≤1 ≥10 ≥2

C III 50≤FEV1<80 <0.70 Severe ≥2 <10 0-1 Very D IV 30

Phenotyping from a clinical standpoint aims to classify patients into distinct sub groups that better define underlying mechanisms and inform therapy. Individuals clustering together with similar mechanistic and/or clinical outcomes should exhibit a similar therapeutic response to drugs. From a research point of view, the grouping of patients should be such that it allows for the enrichment of drug responders in a clinical trial (Ahmed et al., 2018). Identification of different phenotypes will make it easier to identify

who are the key target populations for any given drug and whether this drug should target immune mechanisms or pathways activated in the airway epithelium for example. Phenotyping has been used successfully in asthma where patient populations can be broadly grouped into phenotypes such as Th2 high and Th2 low asthmatics and treatments can be administered accordingly (Sterk and Lutter, 2014). However, it has proved more difficult to distinguish between phenotypes in COPD since COPD is formed from the interplay between several diseases such as emphysema, bronchitis and small airways disease which usually as a result of cigarette smoke exposure in susceptible individuals, increases the rate of annual lung function decline (Han et al., 2010).

COPD mainly develops from cigarette smoking although other factors such as air pollution, the burning of biomass fuels and genetic predisposition can also cause COPD. The prevalence of COPD is similar between men and women in developed countries reflecting the equal rates of smoking between the two sexes (Camp et al., 2009).

The disease generally presents post 40 years of age and is associated with accelerated lung aging (Lozano et al., 2012). The increase in elderly population particularly in developed countries has meant that COPD poses a significant economic burden. As the world’s aging population increases, the global COPD burden is predicted to rise due to continued exposure of an increasing geriatric population to COPD risk factors including cigarette smoking and air pollution (Lozano et al., 2012).

1.1.2 COPD Pathophysiology Chronic inflammation due to the exposure of the airway epithelium to chronic insults from tobacco smoke, pollution and/or infectious agents results in the narrowing of the lumen and emphysematous destruction of the lung parenchyma. Repeated damage to the epithelium together with chronic infections trigger the release of inflammatory cytokines by epithelial cells that cause the infiltration of innate and cells of the adaptive immune system such as, natural killer cells, neutrophils, macrophages, and T- and B-cells respectively into the airway. Immune cells further release cytokines and other inflammatory mediators perpetuating the cellular inflammation (Barnes, 2008, Barnes, 2014).

The intensity of chronic inflammation increases with increasing severity of COPD and is accompanied by structural changes such as goblet cell hyperplasia, ciliated cell hypoplasia, smooth muscle thickening, and extracellular matrix thickening (ECM) (Annoni et al., 2012, Kranenburg et al., 2006, Michaeloudes et al., 2017, Jing et al., 2018). Extra mucous deposition into the lumen further narrows the airway increasing resistance and limiting airflow. Lack of mucocilliary clearance (MCC) and increased cellular damage in the airway results in increased bacterial infection and colonisation. Chronic bronchitis, increased mucous production, is often observed in patients with COPD (Taylor et al., 2010).

Alveolar attachments to the small airways serve to re-open the airways during breathing. Proteolytic enzymes including elastase and metalloproteases are secreted into the alveoli by activated immune cells such as neutrophils and macrophages. These proteases destroy the alveolar attachments and cause the loss of elastic recoil (lung compliance) and the generation of large air pockets in the parenchyma that lead to luminal collapse that further limits airflow (Figure 1.0.0) (Barnes et al., 2015).

Figure 1.0.0-Emphasymatic destruction of alveoli: A Alveoli of healthy patient where airway is held open by elasticated alveolar attachments creating a large empty lumen with mucous coated epithelial lining, B Destroyed alveolar attachments, chronic inflammation, matrix thickening and hyper-mucous secretion into the small airway results in the dramatic shrinkage of luminal space. Adapted from (Barnes et al., 2015) with permission. 30

1.2 Lung Morphology and Development The main purpose of the lung is to facilitate gas exchange, to enable this the lung is composed of a vastly vascularised branching network of tubes called bronchi, that narrow into bronchioles and open into air sacs known as alveoli. The healthy airways are composed of highly quiescent tissue containing small numbers of progenitor cell populations that differentiate to maintain and regenerate a variety of cell types in response to tissue damage (Ahmed et al., 2018). Communication between these resident immune and structural cells and progenitor cells is essential for normal airway homeostasis. Dysregulation of this cell-cell communication and correct cell differentiation during tissue damage and repair leads to the changes in the airway observed in COPD (Ahmed et al., 2018). The pathways that are used to regenerate the structures of the airway are conserved throughout development and into adulthood (Ahmed et al., 2018). Therefore, it is important to understand what governs these developmental processes in order to develop pharmacological agents to better repair or even regenerate the lung after acute or sustained injury.

1.2.1 Overview of Lung Development Airway development occurs through five highly regulated stages (Figure 1.2.1). Definitive endoderm (DE) forms after the appearance of the primitive streak during gastrulation. DE forms a tube with cells that express the pluripotency marker SOX2 and gives rise to the foregut endoderm (AFE). Enhanced localised NKX2-1 expression leads to the out branching of two lung buds at ~day 28 which are surrounded by mesoderm and the vascular plexus ventral wall of the AFE whilst the anterior foregut septates into tracheal and oesophageal tubes (Schittny, 2017). The outgrowth of these buds into a highly ordered branched network is governed by a complex system of genes that coordinate mesenchymal-epithelial signalling. Lung buds branch to give rise to the bronchi by the end of the pseudoglandular stage at ~6 months resulting in a lung structure composed of a complex network of airways surrounded by vasculature (Schittny, 2017). Terminal bronchioles and respiratory bronchioles are formed in the canalicular stage. Bronchioles continue to be expanded distally and primitive alveoli are formed during the saccular stage. Alveoli continue to develop during the alveolar stage and further expand their surface area postnatally (Figure 1.2.1) (Morrisey and Hogan, 2010, Ahmed et al., 2018, Herriges and Morrisey, 2014).

This thesis will focus on the mechanisms occurring at the pseudoglandular stage of lung development.

Figure 1.2.1-Stages of lung development: The lung develops through 5 distinct stages that allow for the formation of the different structures of the lung. Image with permission from http://www.embryology.ch.

1.2.2 Branching Morphogenesis The bronchial tree forms from iterative rounds of bifurcation from the trachea to the alveoli at the distal tips in a process termed branching morphogenesis during the canalicular stage of development (Figure 1.2.1 and 1.2.2). Proper differentiation of airway smooth muscle (ASM) and airway epithelial cells (AEC) requires both the presence of mesenchymal and endodermal protein cross talk. Major protein mediators of the establishment of the bronchial tree are sonic hedgehog (SHH), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and wingless-related MMTV (mouse mammary tumour virus) integration site (WNT), retinoic acid (RA) and transforming growth factor (TGFβ) families (Ludtke et al., 2016).

FGF10 protein is crucial for branching morphogenesis since FGF10-/- rodents show branching agenesis of the lung whilst over expression of FGF10 protein results in overly abnormal branched lung tubules (Min et al., 1998). Other FGFs such as FGF7 and FGF9 are also expressed at this stage of development (Volckaert and De Langhe, 2015). FGF7 protein acts through FGFR2b receptor but promotes epithelial cell 32

proliferation at tubule tips rather than branching (Zhou et al., 1996). FGF9 signalling from the mesothelium has been shown to regulate mesenchymal wnt2a gene expression as well as epithelial FGF9 protein expression, which regulates the expression of the WNT inhibitor Dickkopf 1 (DKK1).

Figure 1.2.2-Tracheal tube morphogenesis from the anterior foregut of mouse embryo: Timed expression and accumulation of protein transcription factors such as NK2 homeobox 1 (NKX2.1) ventrally and expression and SRY (Sex Determining Region Y)-box (SOX)2 dorsally as well as protein transcription factors such as wingless-type MMTV integration site (WNT)s, fibroblast growth factor (FGF)s and bone morphogenetic protein (BMP)s regulate the budding and formation of the early tracheal tube from the oesophageal tube (Goss et al., 2009, Serls et al., 2005), A and B, Timepoints in the septation of tracheal tube to the formation of airway precursor buds, C, Expression of Sonic hedgehog (Shh) in lung tubules, D. Image with permission from (Morrisey and Hogan, 2010). Abbreviations defined as: catenin beta (Ctnnb)1 . forkhead box f (Foxf) 1, glioma associated homologe (Gli) 1 and 3, homeobox (HOX), T-box 4 (Tbx4), transforming growth factor β (Tgfbeta), retinoic acid (RA). FGF10 protein is expressed by the mesenchyme at the distal tips of the nascent blind ended tubules and acts through its receptor FGFR2b. FGF10 localises in specific locations of branchpoints, causes outgrowth of the distal tip resulting in bifurcation and branching (Bellusci et al., 1997) by stimulating the phosphorylation of tyrosine 734 on FGFR2b. This induces Phosphatidylinositol-4,5-Bisphosphate 3-kinase (PI3K) and SH3 (SRC Homology 3) domain binding protein 4 (SH3BP4) recruitment to the receptor complex which allows FGFR2b to be internalised and recycled (Volckaert and 33

De Langhe, 2015). This process highlights the role of the PI3K-AK transforming (AKT) pathway in promoting epithelial cell migration and branching. FGF7 differentially stimulates the phosphorylation of the FGFR2b phospholipase C binding site and interrupts the binding of PI3K and ultimately causes the degradation of FGFR2b and the switching from cell migration to proliferation (Volckaert and De Langhe, 2015). Branching morphogenesis is tightly regulated and is not continuous. After branching, the process may be halted for cellular reorganisation and division before proceeding. The control of branching is achieved by the reciprocal actions and cross talk between different cell layers within the elongating tubule (Volckaert and De Langhe, 2015). Mesenchymal WNT2a signalling through β-catenin regulates FGF10. FGF10/FGFR2b binding in the epithelium causes the release of epithelial cell β-catenin, which in turn regulates the expression of FGFR2b protein receptor and therefore regulates FGF10 protein signalling to the epithelium. FGF10 signalling also results in epithelial SHH signalling, which in turn activates mesenchymal WNT2a signalling thus negatively regulating FGF10. Furthermore, mesenchymal FGFR2b protein receptor is regulated by WNT2 and WNT5a which govern mesenchymal and epithelial proliferation, Figure 1.2.3 (Morrisey and Hogan, 2010, Volckaert and De Langhe, 2015). Epithelial WNT7b has been shown to mediate epithelial β-catenin and FGFR2 protein expression and signals to the mesenchyme where it promotes cell proliferation and maintenance of epithelial and airway smooth muscle (ASMC) progenitor phenotypes (Figure 1.2.3) (Cohen et al., 2009, Wang et al., 2005).

Figure 1.2.3-Epithelial-stromal cell signalling during branching morphogenesis: Protein signalling between the epithelium and the mesenchyme pattern cellular development in the epithelium and the mesenchyme. When this signalling is inhibited or upregulated, ordered branching morphogenesis leads to a quiescent arborised airway with different cell populations. Therefore, the environmental niches that are created by cell-cell signalling are crucial for lung formation. Adapted from (Volckaert and De Langhe, 2015) with permission. Abbreviations in artwork defined as: bone morphogenetic protein (BMP) 4, BMP receptor (BMPR) 1a/b, dickkopf (DKK) 1, fibroblast growth factor (FGF) 1/2c, 9 and 10, FGF receptor (FGFR), frizzled (Fzd) 1 and 10, NMYC proto-oncogene protein (N-myc), patched (Ptch), sonic hedgehog (SHH) wingless-related MMTV (mouse mammary tumour virus) integration site (WNT) 2a and 7b. FGF10 induction of epithelial β-catenin mechanism also specifies the epithelial cell lineage by inducing SRY box 9 (SOX9) and BMP4 expression: the latter inhibiting SRY box 2 (SOX2) to maintain the AEC progenitor population. BMP4 antagonises epithelial cell proliferation in the distal tip of the lung tubule and regulates epithelial AEC progenitor cell outgrowth but is controlled by its own inhibitor Noggin. As the tubule tip extends, FGF10 signals decrease in the proximal epithelium and SOX2 is no longer inhibited and AECs progenitors begin to differentiate into tumour protein 63 (P63+) basal epithelial progenitors which may become further specialised into secretory or non-secretory epithelial cells (Morrisey and Hogan, 2010, Volckaert and De Langhe, 2015) (Figure 1.2.4). At the distal end of the lung tubule, epithelial cells retain their SOX9/inhibitor of DNA binding 2 (ID2) identity and give rise to alveolar progenitors once tubule elongation is concluded.

Figure 1.2.4-Lung tubule extension: Lung tubule extension may be epithelial driven and relies on epithelial-mesenchymal cell-ECM signalling. Mesenchymal wingless-related MMTV (mouse mammary tumour virus) integration site (WNT)/β-catenin signalling stimulate fibroblast growth factor 10 (FGF10) secretion. FGF10-bonemorphogen protein 4 (BMP4) axis has been identified as important for the extension of the lung tubule whilst SRY (Sex Determining Region Y)-box (SOX) 2 and 9 expression controls the maturation of epithelial precursors. Adapted from (Volckaert and De Langhe, 2014).

As well as epithelial lineage specification, FGF10/β-catenin levels also specify mesenchymal ASMC differentiation. Mesenchymal progenitors take more proximal positions as the tubule extends and encounter epithelial BMP4+ and SHH+ cells, which drive ASMC differentiation and negatively regulate FGF10 respectively (Mailleux et al., 2005). The mesenchymal progenitor-ASMC differentiation is known to be induced by WNT2 activation of myocardin and myocardin like 2 (MKL2) (Goss et al., 2011). The interaction between the forming epithelium and mesenchyme is mechanically patterned by the basement membrane (BM). The stiffness of the BM scaffold is important as it directs cells to the right location for branching. Fibronectin has been observed accumulating in areas where branching will occur capping the tubule separating the endodermal and mesenchymal layers. Cells reorder the ECM at distal tips into a porous matrix through protease activity to allow for branching and tubule extension (Harunaga et al., 2014).

1.2.3 The Extracellular Matrix (ECM) The ECM is a 3D structural mesh of proteins, glycoconjugates, carbohydrate polymers and proteoglycans that specify the mechanical properties of the lung airways, such as visco-elasticity, stiffness and thereby deformability. In addition, it is also a major source of signalling between stromal cells of the mesenchyme and the epithelial cells of the endoderm during branching morphogenesis and lung homeostasis. Loss of function studies have shown that ECM depletion during development results in embryonic lethality (Miner et al., 2004, Alpy et al., 2005, Alexopoulos et al., 2009, Hall et al., 2007, Trinh and Stainier, 2004). The ECM occupies a considerable volume of the extracellular space within the lung. Polysaccharide rich proteoglycan (hyaluronan)s, glycoproteins, glycosaminoglycan (GAG)s and structural glycoproteins form a highly hydrated ground matrix hydrogel, to which collagens and elastin fibre proteins, that provide the tissue with biomechanical and visco-elastic properties, are embedded (Figure 1.2.5) (Dunsmore and Rannels, 1996).

Figure 1.2.5-Proteins in the extracellular matrix (ECM): Different structural proteins are found in the ECM. The types of structural proteins and their manner of associations forms the interstitial and basement ECM. These have different biophysical and chemical properties and are involved in the attachment, signalling and maturation of different cell populations. Abbreviations in artwork defined as: glycosaminoglycan (GAG), heperan sulphate proteoglycans (HSPG) and proteoglycan (PG).

The structure of this matrix is particularly important, as the loss of tensile and visco-elastic properties as well as the thickening and compositional change of protein types or concentrations in the airways are pathological symptoms of multiple respiratory diseases such as COPD and idiopathic pulmonary fibrosis (IPF). The influence of the ECM and its correct structural assembly is crucial for the formation of lung organs. There are two broad classes of ECM; interstitial connective tissue that gives structural support by allowing cell-cell attachments and the BM; a specialised

layer of ECM underlying epithelial cells that separates the stroma from the epithelium (Dunsmore and Rannels, 1996). The BM is comprised of a collagen IV lattice that binds laminin, heparan sulphate proteoglycans (HSPG), nidogen and proteoglycans (Figure 1.2.6) (Siegel et al., 2014). BM regulates adhesion, cellular differentiation, migration, proliferation and survival via integrin attachments to cellular receptors and through the release of growth factors sequestered in BM such as epidermal growth factor (EGF), FGF and WNTs (Beqaj et al., 2002).

Figure 1.2.6 Basement membrane architecture self assembly: Helical type IV collagen chains form the lattice through association via their N and C terminals. Secondary polymer network is achieved by the weaving of laminin through the collagen lattice and cross bridges formed by nidogen bind laminin to collagen IV for structural stability. Herperan sulphate proteoglycans (HS-PG) binds to laminin and collagen via glycosaminoglycan (GAG) chains. Dimers and oligomers are stabilised through core protein interactions of perlacan. The structure can easily be modified according to the tissue specific requirements during development, repair etc. Epithelial cells anchor to basement membrane through laminin-integrin interactions. Adapted from (Siegel et al., 2014) with permission. 1.2.3.1 ECM Proteases ECM proteins are cleaved by proteases to remodel ECM structure and to release growth factors and cytokines bound stored in the ECM. The reorganisation of ECM components is crucial for proper lung development and repair (Coraux et al., 2008). Matrix metalloproteinases (MMPs) are the major enzymes involved in ECM cleavage (Beqaj et al., 2002, Miletti-Gonzalez et al., 2012). MMP activity in the healthy adult lung is low. However, the concentration and activity of MMPs increases in inflamed, 38

diseased and injured tissue or during development when constant ECM remodelling is required (Figure 1.2.7). MMPs are soluble and are secreted in a cell membrane bound state and released into the extra cellular space as zymogens which are activated when required through cleavage by other MMPs or by serine proteases. MMPs are regulated through MMP inhibitors such as tissue inhibitor of metalloproteinases (TIMPs) to prevent excessive tissue degradation (Masumoto et al., 2005). ECM remodelling during development is controlled by interplay between MMPs proteins and their inhibitors (Figure 1.2.7). Studies have shown that between weeks 9-42, MMP1 and 9 along with TIMP-1, 2 and 3 are expressed in epithelial cells whilst MMP1, 2 and 9 along with TIMP-2, and 3 are expressed by the endothelium and mesenchyme (Masumoto et al., 2005). Interestingly, genetic and immunohistochemical studies have shown that MMP-1, 2, 8, 9 and 12 proteins are associated with COPD. MMP1 and 2 are highly expressed in epithelial and alveolar regions with MMP1 also being found within the lung parenchyma. MMP8 and 9 are localised to neutrophils and their expression is increased along with that of TIMP1 in broncho-alveolar lavage fluid (BAL) and sputum of emphysema and COPD patients (Russell et al., 2002a, Betsuyaku et al., 1999, Finlay et al., 1997). MMP2 and 12 are increased in alveolar macrophages and have high elastolytic activity in tobacco smoke induced COPD patients (Russell et al., 2002b). Overexpression of MMPs can have devastating effects as seen in COPD where the increased degradation of alveolar attachments to the airways causes luminal collapse leading to airway narrowing (Miletti-Gonzalez et al., 2012, Babusyte et al., 2007).

Figure 1.2.7 Matrix metalloproteinases (MMPs): The table shows the different types of matrix metallo proteinase (MMP) and their various roles during lung cell homeostasis. MMPs such as MMP2 are used to create tracts during collective migration whereas MMP9 is active during cellular repair and is upregulated in COPD. Maternal smoking upregulates different a disintegrin and metalloproteinase (ADAM) in the lungs of neonates. Adapted from (Bonnans et al., 2014) with permission.

1.2.3.2 ECM Proteins and Lung Branching The highly dynamic outgrowth of the nascent bifurcating tubule is reliant upon the structural support of the ECM to form and hold its shape. Continual remodelling through ECM cleavage and deposition is crucial to facilitate mesenchymal- endodermal crosstalk. This coordinates migration of cells to the correct location enabling clefts to form in the invading branch. This process involves epithelial buckling in the distal tubule and the differentiation of progenitor populations into correct cell types within the FGF10-/- proximal section (Bonnans et al., 2014). Several proteins play a crucial role in controlling cell behaviour in organogenesis and some of these are discussed below.

β-1 Integrin Receptors

Several studies have shown that β-1 integrin receptors (Figure 1.2.8) on cell surfaces are absolutely necessary for the proper branching of the airways and their subsequent differentiation and alveolarization. The major integrin receptors involved in cell-ECM interactions during lung development are α3β1, α6β1 and α6β4 (Georges- Labouesse et al., 1996, De Arcangelis et al., 1999, Kim et al., 2009). α3 and α6 deletion leads to a hypoplastic lung in mice with the α3 deletion causing failure of immature tubules to develop into mature bronchioles (Chen and Krasnow, 2012, Plosa et al., 2014).

β-1 integrin regulates important processes in the developing mouse lung. Epithelial deletion of β-1 integrin results in embryonic lethality, abnormal branching, reduced alveolarization, abnormal alveolar differentiation (type I and II pneumocytes were large, cuboidal and had enlarged nuclei) and increased lung inflammation (Chen and Krasnow, 2012, Plosa et al., 2014).

Heparan sulphate (HS)

HS belongs to the glycosaminoglycan (GAG) family of linear polysaccharides, is found on cell surfaces as well as the ECM and is crucial for cell-cell signalling and cell- matrix interactions. It is formed from alternately repeating glucosamine-uronic acid residues attached to a proteoglycan core to make glycosaminoglycans (GAG). HS binds covalently to polysaccharides forming HS proteoglycans (HSPG) such as perlecan found in the basement membrane and syndecans found at cell surface 41

membranes. GAG chains in HS are polyionic where negative charges interact with cations such as Na+ and repulse negatively charged molecules leading to a osmotically hydrated porous gel (Hardingham and Fosang, 1992). GAG chains can be highly modified to suit the requirements of the local cellular niche through the modifications of GlcNAc/GlcUA residues via O-sulfation by sulfotransferases or remodelled through the selective removal of O-sulfates on glucosamine residues by endosulphates secreted at cell surfaces to form highly or lowly sulphated HSPG respectively (Ai et al., 2003, Ohto et al., 2002).

Figure 1.2.8 - Growth Factor and heparin sulphate (HS) interactions: HS proteoglycans (HSPGs) bind a variety of growth factors forming reservoir (a) in addition to binding to cell surface receptors such as integrins (b) and for growth factors (c). HSPG degradation enables the release of growth factors in a concentration-dependent manner making HSPGs key players in growth factor signalling.

HS is acidic and binds basic growth factors such as FGF, BMP, SHH and WNT proteins required for branching morphogenesis (Section 1.2.2, Figure 1.2.6). This provides a reservoir of soluble growth factors within the matrix that can be released in a temporal and tissue specific manner (Asada et al., 2009, Fuerer et al., 2010, He et al., 2017, Hu et al., 2009). For example, although HS binds FGF causing protein inactivation and sequestering (Izvolsky et al., 2003, Lord et al., 2014), active FGF can 42

be released by heparin and heparinases (Izvolsky et al., 2003). However, FGF cannot bind to its receptor without further association with HS polysaccharide side chains. Syndecan on the cell surface acts as a co-receptor to aid in the binding of FGF to its receptor (Rahmoune et al., 1998). Lung explants lacking HS display neonatal lethality (Habuchi et al., 2007).

Research from lung explants has shown that a thin layer of low O-sulfated porous HS with high affinity for FGF10 is secreted by the mesenchyme at tubule budding sites while high O-sulfated HS with low affinity for FGF10 is secreted adjacent to proximal part of the tubule (Patel et al., 2008). This suggests a role for the ECM in controlling FGF10 release during tubule elongation. Embryonic lung explants treated with over O-sulfated heparin interfere with epithelial cell-FGF10 binding and disruption of the FGF10-BMP4 axis which controls lung branching (Izvolsky et al., 2003). However, FGF10 is blocked by netrin around the neck of branching tubules inhibiting further budding and ensuring fractal bifurcation (Liu et al., 2004). The location of the ECM may also further trigger progenitor cell differentiation by cues from ECM bound growth factor at the local niche (Liu et al., 2004). Overall, it is evident that HS are vital for the formation of the lung and for cell-cell signalling during and after lung development.

Laminin

Laminins are heterotrimeric glycoproteins with α, β and γ chains which are only found in the basement membrane. 15 isoforms have been shown to form via the association of 5α, 3β and 3γ laminin chains. α, β and γ chains intertwine forming the long arm of laminins with three shorter arms formed from the outward extension of each chain from the long arm into a cruciform shape (Figure 1.2.7). The C-terminal domain of the long arm binds 5 Laminin G domain-like (LG) modules (LG1-5) where modules LG1 and 2 are the integrin binding domains and LG4-5 bind heparin binding cells to the ECM. These modules can be cleaved by proteases to release cells from the ECM (Nguyen and Senior, 2006).

All laminin α chains are expressed during lung development. α1, 3 and 5 chains are synthesised by epithelial cells whilst mesenchymal cells produce α2 and 4 isoforms. α3, 4 and 5 isoforms predominate in the adult lung while the other two isoforms are mainly found during lung development. α1, 2, 3 and 5 isoforms co-localise

to the epithelial basement membrane whilst α4 is localised to the smooth muscle pericellular membrane lining the epithelium (Figure 1.2.9) (Pierce et al., 2000).

Figure 1.2.9 Structure of laminin: The laminin structure is characterised by 4 binding sites denoted α, β, γ and the integrin binding site whose tails form a triple coil stalk. Adapted from (Nguyen and Senior, 2006) with permission. Abbreviations in artwork defined as: laminin L4 domain (L4), laminin LF domain (LF), laminin G domain-like (LG) 1, 2, 3, 4, or 5, laminin N terminal (LN) and laminin-type epidermal growth factor-like (LE) a, b, or c.

Laminins α1 and α2 are the most extensively studied laminins in branching morphogenesis (Pierce et al., 2000). Schuger et al. and colleagues decreased branching morphogenesis in embryonic mouse lung explants using monoclonal antibodies to the α1, β1 and γ1 chains of laminin 111. Loss of epithelial polarisation was observed upon the blockade of Laminin 111 polymerisation resulting in malformed basement membrane and reduced branching in lung explants (See section 1.2.4) (Schuger et al., 1996). Mesenchymal cells beneath the epithelial cell layer 44

differentiated into smooth muscle while the remaining mesenchymal cells became rounded and did not express any smooth muscle markers (Schuger et al., 1996). The α2 isoform of laminin is integral to the laminin mediated downregulation of RhoA activity during mesenchymal cell differentiation (Schuger et al., 1990b).

Foetal cell organotypic cultures have been used to demonstrate the role of laminin 111 in epithelial-mesenchymal reorganisation (Schuger et al., 1990a). HSPG-Laminin 111 binding is also essential for cell polarisation and lumen formation (Schuger et al., 1996). Although embryos express laminin α5 from early development to adulthood, explant cultures have shown that laminin α5 is not involved in branching morphogenesis (Nguyen et al., 2002, Pierce et al., 2000).

1.2.4 Luminogenesis Lung bud outgrowth is followed by the polarisation of the central tip cell population leading to the formation of a lumen that internally expands with the branching tubules. Lumens are a continuous hollow inner space that run the length of branched tubular organs and are conserved across different species such as mouse and Drosophila (Goldstein and Macara, 2007, Tepass, 2012). Epithelial cells line the lumen and regulate the diffusion of gasses, fluids and other substances into and out of the luminal space (Rackley and Stripp, 2012). During luminogenesis, lumens form by the acquisition of epithelial cell apical-basal polarity where cells define their apical surface by discerning the physiological differences in their microenvironment in three dimensions. These interactions occur between neighbouring cells and the ECM (Rodriguez-Boulan and Macara, 2014). At the end of this process, epithelial plasma membranes acquire apical domains that face the lumen and basolateral domains that interact with the ECM (Rodriguez-Boulan and Macara, 2014). The process of epithelial apical-basal polarisation is conserved in other tubular organs such as the vascular tubes, kidney tubes and, mammary glands (Bryant and Mostov, 2008). Several diseases can arise from the failure of epithelial cell polarisation including COPD (Nishioka et al., 2015).

1.2.4.1 Forming an Apical-Basal Identity: Cell-Cell and Cell-ECM Interaction Epithelial cells define paracellular boundaries by forming tight and adherens junctions. Tight junctions are formed through the interactions of claudins, occludins and junctional adhesion molecule (JAMs) proteins at paracellular junctions which form 45

a seal between cells (Ahn et al., 2016). Tight junctions bind plasma membranes of adjacent cells acting as a barrier to uncontrolled paracellular diffusion of lipid and protein molecules. Adherens junctions are formed from the association of cadherins and nectins, are basal to tight junctions and provide mechanical stability by linking to the actin cytoskeleton (Suzuki et al., 2001, Lin et al., 2000). The level of cadherin expression modulates cortical actin cytoskeletal contraction and therefore cellular tension. Studies have shown that increases in cadherin can result in stiffer cells due to increased actinomysin contraction. Stiffer cells have been shown to translocate to the centre of the cellular mass during Mardin-Darby Canine Kidney (MDCK) cells sphere formation (Martin-Belmonte et al., 2008, Martin-Belmonte and Rodriguez- Fraticelli, 2009). Cells were unable to sort and form a lumen when the linkages between cadherin and the actin cytoskeleton were removed. These studies highlight the importance of paracellular proteins and boundary definition in luminogenesis.

Epithelial cells bind to the ECM via α and β integrins (Levi et al., 2006). As well as embryonic lethality observed in β1-integrin knockout mice, the laminin-β1-integrin- ras-related C3 botulinum toxin substrate 1 (Rac1) module has been shown to control apical basal polarisation in MDCK 3D cultures. β1-integrin knock down caused incorrect activation of RhoA-ROCKI-myosin II activation resulting in the abnormal orientation of the apical surface to the lateral wall of the cells (Yu et al., 2008a).

Alongside cell integrin expression, the arrangement of integrins for the transmission of ECM-cell mechanical signals is crucial for luminogenesis. Studies have shown that a stiff ECM promotes integrin clustering leading to the activation of Rho and assembly of focal adhesions (Yeh et al., 2017). Due to the lack of flexibility of a stiff ECM, higher tensional forces develop at focal adhesions which are transmitted to the cytoskeleton via vinculin (Grashoff et al., 2010).

Apical polarity is formed through the expression of three canonical polarity complexes; crumbs (CRB), partitioning-defective (PAR) and Scribble (SCRIB) complexes (Campanale et al., 2017). Apical-basal polarity program is complex and requires the correct triggering of multiple processes including the formation of apical junctional complexes formed by tight and adherens junctions, basolateral complexes and apical complexes. The triggering of this precise sequence of events has not yet

been elucidated, and the initial arrival proteins at the different domains is not known. However, without the establishment of apical basal identity luminogenesis does not occur (Overeem et al., 2015, Bryant et al., 2014).

1.2.4.2 Lumen Formation Once apical-basal polarity is established, further specialisation of the apical membrane through vesicular trafficking governed by polarity complexes is required for luminogenesis to occur in a process termed hollowing. De novo lumen formation of mammalian epithelial cells begins with the delivery of vesicles containing apical proteins to a discrete landmark between neighbouring cells termed the apical membrane initiation site (AMIS). The AMIS is formed by a polarity complex consisting of partitioning defective 3 (PAR3), atypical protein kinase (aPKC) and the exocyst subunit exocyst complex component 4 (EXOC4) and the adherens complexes E- cadherin, occludin and exocyst complex component 5 (EXOC5).

Figure 1.2.10-Luminogenesis via vesicular trafficking in MCDK cysts (Hollowing): Accumulation of E-cadherin, occluding and exocyst 5 (EXOC5) forms an apical membrane initiation site (AMIS) and polarisation initiates (A). RAB11A Member RAS Oncogene Family (Rab11a), EXOC5, syntaxin3 mediated atypical recycling endosomes (ARE) delivery of CRB and podocalyxin to cell-cell contacts forms a pre-apical patch. Partitioning defective (PAR3) and atypical protein kinase (aPKC) at the pre-apical patch form immature junctional complex, B, mature cyst forms containing polarised cells with adherens and tight junctions with fluid filled lumen, C. Vesicle delivery to the AMIS is driven by a RAB-dependent cascade. RAB11a positive vesicles become enriched with CRB3a, podycalyxin/gp135 and other apical proteins such as mucin (MUC)1 (observed in mouse pancreas in vivo) post cellular mitosis (Bryant et al., 2010). Rabin8 guanine exchange nucleotide factor (GEF) is recruited by RAB11a, which in turn activates RAB8a/b at the AMIS. Vesicle fusion to the AMIS is likely to occur via SNARE proteins (an acronym derived from SNA (Soluble

NSF (N-ethylmaleimide-sensitive factor) Attachment Protein) receptor) are a large protein complex consisting of at least 24 membrane proteins) mediated by syntaxin 3. RAB8/11 vesicles at the apical pole activate cell division cycle (CDC)42 which forms the CDC42-PAR3-aPKC complex, which together with phosphatidylinositol-

4,5-bisphosphate (PIP2) and annexin (ANX)2 controls exocytosis (Figure 1.2.10) (Bryant et al., 2010).

The continuous delivery of complexes matures the AMIS into a preapical patch. Partitioning defective 3 (PAR3)- membrane palmitoylated protein 5 (PALS1) association is disrupted by PAR3 phosphorylation which is restricted to junctional complexes allowing for the formation of CRB-PALS1-PatJ complex that also mediates the dissociation of PAR3 from PAR6-aPKC (Walther and Pichaud, 2010).

Once a lumen has formed, epithelial cells divide in the same plane and the polarised architecture of epithelium is maintained. CDC42-Par3 recruitment of aPKC at the apical surface allows for the phosphorylation and lateral geniculate nucleus (LGN)- nuclear mitotic apparatus protein 1 (NUMA) complex by aPKC resulting in its relocation to the lateral domains inhibiting division in the apical domain (Ajduk and Zernicka-Goetz, 2016).

Another prevailing theory for luminogenesis termed cavitation is derived from early experiments in mouse embryos and is the predominant mode for lumen generation in mammary and salivary gland development (Jaskoll and Melnick, 1999, Jaskoll et al., 2002, Debnath and Brugge, 2005, Mailleux et al., 2008). Cavitation is initiated when cells aggregate into spheroid clusters. The outer cell population of the spheres become polarised due cell-ECM interaction and signalling. The central spheroid cell population remains unpolarised and becomes detached from the outer cell populations and undergo apoptosis via a B-cell lymphoma 2 (BCL-2), BCL2-like protein 11 (BIM) and SH3-Domain GRB2 Like Endophilin B1 (BIF) mediated proapoptotic pathway (Figure 1.2.11) (Humphreys et al., 1996, Debnath et al., 2002). Autophagy pathways in central cell population are also upregulated indicating that other methods of luminal clearing may also be present.

Figure 1.2.11-Luminogenesis by cavitation- Cell aggregation and division, A, outer cell populations become polarised due to ECM interaction whilst central cell populations undergo apoptosis or autophagy due to anoikis, B, cells continue to divide in the outer plane forming an inner lumen, C. Although two distinct types of luminogenesis have been found, it is likely that both methods occur in organogenesis. Experiments from MDCK cysts have shown that in stiff ECM such as collagen where rapid polarisation of cells cannot occur, luminogenesis occurs via cavitation as the outer spheroidal cell population must synthesise their own matrix before cell polarisation can occur. However, in a more differentiated softer matrix such as matrigel, cells rapidly polarise, and hollowing methods are utilised (Martin-Belmonte et al., 2008, Humphreys et al., 1996).

1.2.4.3 Lumen Expansion Luminal expansion of the tubule occurs through increases in hydrostatic pressure by increase of fluid in the lumen. Hydrostatic pressure inhibits luminal collapse in the early stages of development as the requisite supporting structures such as cartilage rings, smooth muscle, parenchymic ECM and the alveolar attachments required to open the lumen are still in development (Hooper and Harding, 1995).

Channels such as Na-K-adenine triphosphatase (ATPase) and cystic fibrosis transmembrane conductance regulator (CFTR) ion pumps mediate the influx of fluid and chloride ions into the central lumen of MCDK cysts (Yang et al., 2008, Bagnat et al., 2007, Kesavan et al., 2009). Mutations in CFTR greatly inhibit luminal expansion for example hyperactivation of CFTR channels in intestinal organoids by forskolin treatment leads to rapid expansion of organoid lumens. The same expansion is not observed in intestinal organoids formed from cystic fibrosis patients where CFTR is perturbed (Boj et al., 2017, Dekkers et al., 2013). During organogenesis in multiple 49

organs, multiple micro lumens coalesce to form one central larger lumen in a claudin mediated permeability through tight junctions. The inhibition of claudins results in the formation of multiple lumens (Bagnat et al., 2007, Gutzman and Sive, 2010).

The maintenance of apical basal polarity is crucial for the formation of a barrier that controls the passage of particulates within tubular organs such as the lung airways. Diseases such as asthma and COPD have been shown to develop from loose paracellular connections that allow the uncontrolled passage of allergens pollutants, pathogens and other harmful particulates that can cause damage to the underlying cell populations (Shaykhiev et al., 2011, Hogg and Timens, 2009, Holgate et al., 2003). The constant assault of cells can lead to irreversible damage leading to incorrect cellular differentiation and repair as well as increased matrix deposition that can alter the stiffness of the underlying lamina (Shaykhiev et al., 2011, Hogg and Timens, 2009, Holgate et al., 2003).

1.3 Lung Epithelial Cell Populations The continued growth and elaboration of the lung tubule gives rise to several populations of progenitor cells that differentiate and form the different somatic cell layers that eventually form the adult airway lumen (Figure 1.3.1). Progenitor cells of the adult lung are maintained under tight control with an increase or decrease in progenitor populations resulting in defective lung repair or respiratory disease (Stripp, 2008, Spella et al., 2017). Studies in mouse lung have been the most effective in identifying lung progenitor populations (Leeman et al., 2014). However, the precise set of events that leads to the development of fate committed epithelial cells is unknown.

One model suggests that the acquisition of SOX2+ marker by precursor epithelial cells in proximal positions lining the lumen of the growing lung tube directs cell fate towards either a neuroendocrine (NE) fate where cells express achaete-scute homolog 1 (Ascl1) or a non-NE fate where cells express secretoglobin 1A 1 (Scgb1a1), secretoglobin 3A 1 (SCGB3A1) and secretoglobin 3A 2 (SCGB3A2) (Reynolds et al., 2002, McCauley et al., 2018). Hairy and enhancer of split 1 (Hes1) is upregulated in non-NE and has been shown to inhibit Ascl1 and therefore inhibiting non-NE fate and driving NE cell development although the mechanism for this process is not fully

characterised (Meder et al., 2017). Interestingly, Hes1 expression is controlled by Notch signalling which is required for formation of secretory cells.

Figure 1.3.1-Lung epithelial population: Basal cells and cilia line the airways from the nasal passage till the distal airways. Ciliated cells make 30% of the respiratory population. Goblet cells are mainly found in the trachea and bronchi and reduce in number towards the terminal bronchi where more club cells population increases, A, lung progenitors differentiate to make different cells lining the airway during development although precise mechanism is unknown and there are likely other unidentified progenitor populations involved, B. Gene names in artwork defines as: Achaete-scute homolog 1 (Asc1l), calcitonin gene related protein (Cgrp), cytochrome 450 (Cyp450), forkhead box J1 (Foxj1), keratin 5 (Krt5) mucin 5 A/C (Muc5AC), neuro endocrine (NE) cell, secretoglobin 1a 1 (Scgb1a1), sex determining region Y (SRY)-box 2 (Sox2) and tumour protein 63 (P63). Cells further specialise and express transcription factor tumour protein 63 (P63), keratin 5 (KRT5), integrin subunit α 6 (ITGA6) and nerve growth factor (NGFR) becoming basal progenitor cells. One possible mechanism may involve inhibition of WNT protein signalling via DKK1 causing SOX9 inhibition and promoting sox2 gene expression which is known to directly control ΔNP63 expression (see below) (Ochieng et al., 2014). p63 is fundamental to basal cell formation and its ablation in mice results in the loss of basal cell population and the formation of a simple columnar epithelium, whilst ectopic expression causes squamous metaplasia (Daniely et al., 2004). The p63 51

gene has two transcribed isoforms distinguished by TA containing region (TAP63) or the lack of a ΔN region (ΔNP63). The two isoforms are post translationally edited into a further four isoforms each (Warner et al., 2013). The different isoforms have been shown to be expressed at different levels in basal cells of different organs such as ovaries, liver, mammary glands and skin and lungs. ΔNP63 is highly expressed in human and murine lung basal cells and has been linked to a variety of progenitor homeostatic processes (Warner et al., 2013). However, its role in lung basal cell homeostasis is yet to be fully elucidated although studies have shown that a functional interaction between P63 and yes associated protein (YAP) is necessary to maintain the airway epithelium (Mahoney et al., 2014, Zhao et al., 2014). In humans, basal cells line the respiratory epithelium from the nose to the end of the bronchioles and are crucial for the development, maintenance and repair of the pseudostratified epithelium (Boers et al., 1998, Roberts et al., 2018). Basal cells differentiate to form ciliated and goblet cells of the airway epithelium through the tight control of Notch signalling (Gomi et al., 2015, Herfs et al., 2012, Coote et al., 2015). Different Notch proteins and ligands have been shown to promote specific cell populations in the developing lung. For example inhibition of the notch3 gene led to basal cell metaplasia whilst mice with conditional knockout of hes1, recombination signal binding protein for immunoglobulin κ J region (rbpj-κ) or protein O-fucosyltransferase 1 (pofut1) had increased ciliated and club cell numbers and major loss of secretory cells (Mori et al., 2015, Tsao et al., 2011). Conversely, upregulation of notch signalling resulted in the expansion of secretory cells at the expense of secretory cells (Gomi et al., 2015, Tsao et al., 2009). Jagged 1 (JAG1) deletion in mice resulted in decrease of delta like canonical Notch ligand 1 (DLL-1), HES-1 and HES-5 proteins together with goblet cell metaplasia, an increase of ciliated cells and a reduction in club cells demonstrating that JAG1 protein is necessary for mucin inhibition and required for club cell development (Herfs et al., 2012, Zhang et al., 2013). Danahay et al showed NOTCH2 inhibition prevents goblet cells metaplasia in a human 3D organoid culture (Coote et al., 2015). Other cell types that are reliant on Notch signalling are neuro endocrine (NE), ATI and ATII cell differentiation which are beyond the scope of this thesis. Ciliated cells express marker forkhead box (FOX)J1 which is necessary for ciliogenesis (Yu et al., 2008b). Recent studies have shown that FOXJ1 expression 52

occurs late in ciliogenesis indicating that rather than committing basal cells to a ciliated fate, FOXJ1 marks cells already committed to ciliogenesis. Unlike secretory cell fates, there are no current validated pathways that commit basal cells to ciliated cell fates. A recent paper documented a concentration-dependent increase in ciliated cells in response to interleukin (IL)-6 in human bronchial epithelial and murine cell air liquid interface (ALI) cultures. Furthermore, IL-6 increased FOXJ1 expression through the janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 pathway and subsequent down regulation of notch1 (Herfs et al., 2012). The differentiation of SOX2+ cells to different epithelial lineages still needs to be characterised.

1.3.1 Airway Smooth Muscle (ASM) Development The mesenchyme of the lung gives rise to airway and vascular smooth muscle, pericyte, myofibroblast, endothelial and interstitial fibroblast cells. The development of these tissues requires epithelial-mesenchymal signalling just as the development of the epithelium from the endoderm required mesenchymal to epithelial signalling (McCulley et al., 2015, Volckaert and De Langhe, 2015, Shannon and Hyatt, 2004). ASM is found in between the tracheal cartilage rings and band across lower airways that are devoid of cartilage. ASM cells control the diameter of the airways with defects in ASM development or maintenance resulting in respiratory disease such as asthma or COPD where ASM layer is thickened and constricted adding to airway luminal diameter reduction and therefore obstruction (Michaeloudes et al., 2017). In asthma, ASM is hyperresponsive and can constrict to totally close the airway, which if left untreated would result in death (Doeing and Solway, 2013). ASM cell progenitors are derived from a group of mesenchymal progenitor cells found at the outer tip bud of the developing tubule. Lineage tracing experiments have shown that mesenchymal progenitors migrate from the tip to the lung stalk differentiating into elongated ASM cells that band transversely to the growing epithelial tubule (Kumar et al., 2014). ASM cell development is governed by WNT/β-catenin-FGF9 signalling. Mesenchymal WNT signalling is required for FGFR1c/2c generation. Mesothelial and epithelial FGF9 binds to this receptor and has been shown to increase mesenchymal cell proliferation (Yin et al., 2011). FGF9 inactivation in Dermo-Cre mice has shown a

marked reduction in proliferation of mesenchymal cells without affecting branching morphogenesis of the epithelial layer (Cohen et al., 2009). ASM cell fate is adopted by mesenchymal cells in response to mesenchymal β-catenin, which acts together with mesenchymal FGF signalling to stop premature ASM maturation through the control of FGF10 expression and signalling (McCulley et al., 2015, Volckaert and De Langhe, 2015, Shannon and Hyatt, 2004). In addition, BMP4 secreted in response to mesenchymal FGF10 drives ASM differentiation. Mature ASM express the BMP4 inhibitor Noggin which results in tight feedback control of BMP signalling in the distal airways (Mailleux et al., 2005, Ramasamy et al., 2007, Yin et al., 2008).

1.4 Airway Regeneration, Repair and COPD Default cell turnover in the human lung is low compared to other tubular organs but increases in response to acute injury to enable regeneration of the requisite local cellular populations. Basal cells underlie the pseudostratified epithelium and generate cell populations lost due to injury through differentiation and via cross talk with stromal cells such as fibroblasts (McCulley et al., 2015, Volckaert and De Langhe, 2015, Shannon and Hyatt, 2004). Experiments where acute lung injury was induced in mouse models through sulfur dioxide (SO2), naptheline or cigarette showed a basic sequence of events occurs to restore the lung which are described in detail here. Upon injury, inflammatory factors are released recruiting macrophages, neutrophils and STEM/niche progenitor cells to the site of injury. Immune-epithelial-stromal cell communication results in the secretion of soluble factors that restore barrier function through wound closure by epithelial and fibroblast cell spreading and migration. TGFβ signalling promotes trans-differentiation of epithelial cells to mesenchymal like cells during epithelial to mesenchymal transition (EMT) and the initiation of the chronic response. Secreted MMPs allow for remodelling of the ECM to aid in cellular migration division and spreading. Cells undergo mesenchymal to epithelial transition (MET) to restore the epithelium with STEM/niche progenitor cells differentiating to restore local cell populations. Following regeneration of the airway wall, immune cells are deactivated, cellular hyperproliferation decreases, the ECM is restored and wound repair is

resolved (Crosby and Waters, 2010, Adair et al., 2009, Adamson and Bowden, 1975, Ahdieh et al., 2001).

The normal process of lung repair is dysregulated in COPD (Brandsma et al., 2017). The continuous damage caused to the epithelium by cigarette smoke (CS) results in a remodelled airway epithelium with basal and goblet cell metaplasia, ciliated cell hypoplasia, thickened ECM and a high level of inflammatory immune cell infiltration. This is due, in part, to aberrant repair and regeneration mechanisms. Increased goblet cell hyperplasia releases excess mucous into the airway lumen and together with increased inflammation, lack of clearance due to ciliated cell hypoplasia and ECM thickening results in airway obstruction (Jones et al., 2016, Hiemstra and van der Does, 2017).

ECM thickening is caused by the excess release of MMPs and reduced expression of TIMPs during EMT that leads to aberrant ECM turn over, peribronchial fibrosis and excess degradation of the surrounding parenchyma. Enhanced expression of fibroblast factors that may drive EMT such as MMP9, S1004A and vimentin were found in the gaps between degraded reticular BM fragments in smoker and COPD patients (Sohal et al., 2010). Furthermore, CS and nicotine have been shown to drive EMT through the loss of E-cadherin which leads to dysfunctional β-catenin/WNT signalling and the resulting activation of the TGFβ/SMAD, IL-1 and tumour necrosis factor (TNF)-α pathways (Heijink et al., 2016, Gohy et al., 2015).

Loss of barrier function in COPD due to decreased reconstitution of tight junctions is well documented (Hogg and Timens, 2009, Ganesan et al., 2013). CS exposure leads to loss of tight junction proteins due to the reduced expression of junctional genes such as claudins, cadherins, occludins and PAR leading to increased barrier permeability which allows noxious agents such as allergens, pathogens and other pollutants to enter the blood (Hogg and Timens, 2009, Shaykhiev et al., 2011, Heijink et al., 2014).

Lung developmental and homeostatic pathways including histone deacetylase (HDAC)1/2, NOTCH, SHH and WNT, are dysregulated in the lungs of smokers and COPD patients (Ahmed et al., 2018). Epithelial cells from smokers and COPD patients have been shown to express higher levels of NOTCH2 and reduced levels of NOTCH1

and -3 which skews basal epithelial cell differentiation towards generating secretory cells (Section 1.3.0). Basal cell metaplasia also infers that basal progenitor cell regeneration capacity is inhibited or defective in smokers and COPD airways (Staudt et al., 2014). The reason for this lack of regenerative ability is not known. Cardoso et al. have shown that NOTCH3 activation via JAGs results in the differentiation of basal epithelial cells to regenerate the epithelium. Inhibition of NOTCH3, via the γ-Secretase inhibitor (DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), in murine ALI cultures resulted in the expansion of basal epithelial cells (Mori et al., 2015). This was also shown in NOTCH3-/- murine tracheal cells, although these cells could not differentiate implying that differentiation only occurs in the presence of JAG-activated NOTCH3. Therefore, they suggest that although JAG1/2 is continuously expressed, NOTCH is not activated. Once P63 levels reach a threshold due to cellular expansion, NOTCH3 is activated in a select basal cell subpopulation via JAG1/2 and this triggers differentiation through NOTCH1 and -2 (Mori et al., 2015).

Other cell culture experiments have shown that induction of inflammatory mediators such as TNF-α, IL-6, and IL-1β by nuclear factor kappaB (NF-B) in normal human bronchial epithelial cells (NHBE) obtained from healthy smokers resulted in the upregulation of the TA isoform of P63. This suggests a mechanism by which inflammation can modulate airway epithelial differentiation and repair (Herfs et al., 2012). As previously discussed, (Section 1.3.0), how P63 contributes to cellular differentiation is poorly understood. Interestingly P63 has been shown to directly increase JAG1/2 and inhibit proteins such as cyclin dependent kinase inhibitor 1A (P21) (Westfall et al., 2003). Interestingly, increases in P21 are observed in senescent epithelial and fibroblast cells from smokers and COPD patients which suggests a role in premature lung aging (Chiappara et al., 2013).

A major hallmark of COPD is ageing. A prevalent theory of COPD pathogenesis is that continuous exposure to CS causes the small airways to prematurely age due to perturbed repair as a result of increased cellular senescence (Rashid et al., 2018). Cell turnover is an important process during epithelial homeostasis. Though the lung is formed from a quiescent cell population, cell renewal still occurs and a balance between cell death and proliferation is required for the renewal of the airway epithelium under healthy conditions. Exposure of rodents to CS results in the senescence of 56

inflammatory cells, lung fibroblasts and niche progenitor cells which contribute to ineffectual replication and reduced cell turnover (Rashid et al., 2018). A deficiency in cell autophagic and necroptotic pathways also results in a lack of clearance, degradation and recycling of organelles and the persistence of abnormal cells (Chen et al., 2008). The driver for cellular senescence may be cellular damage resulting from increased exposure to reactive oxygen species (ROS) from dysfunctional mitochondria (Boyer et al., 2015).

Premature aging and reduced lung function in COPD may also derive from incomplete development of the lung (Vogelmeier and Bals, 2007). Incomplete lung development is associated with reduced lung function as the normal rate of lung function decline begins from a lower baseline (Boucherat et al., 2016). Factors that give credit to this proposal are studies that show associations between maternal smoking and a 1.5% reduction in FEV1 and 5% reduction in overall expiratory flow (Thacher et al., 2018). Studies on foetal monkeys that were exposed to nicotine in utero, showed altered lung morphology with larger air sacs, increased matrix deposition leading to increased airflow resistance and a decrease in forced mid-expiratory flow (Sekhon et al., 1999, Sekhon et al., 2002). These physiological and morphological features are comparable to those seen in human infants whose mothers smoked (Maritz et al., 1993, Elliot et al., 1998, Lieberman et al., 1992).

Oxidative stress is a key player in the development and pathogenesis of COPD (Rahman and Adcock, 2006). Oxidative stress occurs in all animals that aerobically respire and is required during development as the early embryonic environment is hypoxic (Shinkai et al., 2005). However, excessive chronic oxidative stress due to an oxidant/antioxidant imbalance, can lead to increased cellular damage (Rahman and Adcock, 2006).

.- . Cell exposure to CS leads to the generation of ROS such as O2 and OH that have unstable unpaired electrons that can cause DNA damage and peroxidation of cell plasma membranes, proteins and carbohydrates (Rahman and Adcock, 2006). ROS interferes with cell-ECM attachments by causing integrins to be redistributed to the apical surface of cells and disrupting epithelial barrier function (Gon et al., 2011, Jabbour et al., 1998). Epithelial cells respond to ROS and increased cellular damage

by upregulating intracellular signalling pathways such as the mitogen activated protein kinase (MAPK) and NF-B pathways and enhancing the expression of immune cell mediators including interleukin 8 (CXCL8), granulocyte macrophage colony stimulating factor (GMCSF) and TNFα (Wu et al., 2014). Oxidative stress increases mucin 5A/C (MUC5AC) secretion in the airways through upregulation of the epidermal growth factor receptor (EGFR) (Shaykhiev and Crystal, 2014b, Casalino-Matsuda et al., 2009). EGFR and NOTCH activation have been shown to drive basal cell metaplasia in COPD (Casalino-Matsuda et al., 2009).

Antioxidant defence systems are attenuated in COPD allowing for excess oxidative stress-induced cellular damage. The nuclear factor erythroid derived 2 like 2 (NRF2) pathway as well as phase II detoxifying enzymes are downregulated by ROS accumulation in COPD (Fan et al., 2013, Yamada et al., 2016). Glutathione (GSH) plays a key antioxidant role in airway epithelial cell redox homeostasis. GSH is found in cells whilst glutathione peroxidase is found in extracellular fluid. Glutathione binds to and sequesters oxidised products such as lipid peroxides and stops oxidised metabolites reacting with and damaging cells and tissues (Sthijns et al., 2014). GSH is oxidised to glutathione disulphide (GSSG) which is either reduced back to GSH or excreted. The GSSG/GSH ratio is an important measure of the cellular redox status (Ghezzi et al., 2005, Rahman and Adcock, 2006). GSH is attenuated in COPD epithelial cells and CS exposure causes irreversible modifications to GSH resulting in a depletion of the GSH pool and skewing the GSSG/GSH balance (van der Toorn et al., 2007). Therefore, the reduced antioxidant capacity in COPD results in an excessive response to exogenous and endogenous oxidative stress.

1.5 Current Models of the Airway The development, regeneration, repair and maintenance of the human lung is poorly understood with major gaps in our knowledge of key pathways that regulate processes from stem cell generation to whole tissue organisation. Our understanding of key mechanisms has improved due to the use of different in vivo and in vitro models. However, the area of lung disease needs new approaches or technologies to further aid the discovery of perturbed cellular populations and pathways and to generate targets for novel pharmacological therapies against disease.

1.5.1 Murine Models Mouse studies have provided most of the information for the current understanding of the key airway epithelial developmental and repair pathways. They have been invaluable in lineage tracing studies to determine new potential progenitor cells in response to lung damage (Rawlins et al., 2009a, Rock et al., 2011, Tropea et al., 2012). Few therapies have been developed in mice but currently no better pre-clinical models for testing the systemic effects of pharmacological agents exist (Ghorani et al., 2017).

However, there have been major issues in translating data from mice to humans resulting in the low number of new drugs being approved for COPD (Knight, 2008). Mouse respiratory physiology and epithelial cellular organisation is fundamentally different to that of humans. For example, whilst basal cells line the respiratory tract from the nasal lining to the end of the bronchioles in man, basal cells are missing in the murine trachea (Nakajima et al., 1998, Boers et al., 1998). In addition, mouse airways are less ciliated, have few submucosal cells and no goblet cells compared to human airways. Mice are obligate nasal breathers and do not expectorate sputum and filter cigarette smoke inefficiently (Perez-Rial et al., 2015, Dawkins and Stockley, 2001). The branching of the mouse airway is monopodial with less branching than in human airways and bronchioles, the site of obstruction in COPD, are absent (Chamanza and Wright, 2015, Irvin and Bates, 2003, Walker et al., 2012).

1.5.2 2D Models of Lung Disease In vitro research into human disease processes were originally investigated using immortalised cell lines such as A549 or BEAS2B cells. Early research with these cell lines progressed the understanding of epithelial cell behaviour and have been useful in cancer research. However, by their very nature these cells are abnormal and therefore have limited predictive power. For example, the generation of cell lines involves substantial genetic changes and selection of surviving clones. Therefore, finding adequate models of disease is difficult especially in complex lung diseases such as asthma, COPD and IPF that involve many cell types (Sachs and Clevers, 2014, Reddel et al., 1991). Furthermore, cultured epithelial cells dedifferentiate in monolayer culture losing their cell specificity which is unrepresentative of the in vivo situation.

The air-liquid interface (ALI) culture system allows for the organotypic differentiation of patient-derived primary epithelial monolayers into pseudostratified epithelium and has been widely used. Human bronchial epithelial cells (HBE) are seeded into transwell inserts with a semipermeable membrane that can be fed basally allowing for mucocilliary cell differentiation to occur on the apical surface (Kaartinen et al., 1993). The transwell system has many advantages as cells can be treated apically whilst aerosol, allergen and other particles can be deposited basally (Cao et al., 2018, Gras et al., 2017). Junctional analyses can be performed using transepithelial electrical resistance (TEER) (Boda et al., 2018). ALI cultures have advanced our knowledge of barrier function, response to infection and of epithelial development and airway remodelling in diseases such as asthma and COPD in a reproducible manner as reviewed elsewhere (Mertens et al., 2017b, Mertens et al., 2017a). The monolayer and ALI culture systems have also enabled analyses such as single cell RNA-sequencing methods to be utilised (Ray et al., 2015). In addition, recent studies have attempted to model cell-cell communication through seeding of stromal cells in the apical wells (Malavia et al., 2009).

1.5.2.1 Lung on a CHIP (LOC) Recent advances in microfluidics technology have enabled the creation of biomimetic cell culture devices that have advanced ALI cultures. Chips containing an upper channel the size of a bronchiole were seeded with ATII cells and differentiated as an ALI to ATI cells. The lower channel, separated by a polyvinylidene fluoride (PVDF) membrane, was seeded with endothelial cells to mimic the alveolar-vascular barrier. The model was used to show the effect of mechanical stimulation on nanoparticle uptake and transportation (Huh et al., 2010). Benam et al. later adapted this technique differentiating patient-derived healthy and COPD bronchial epithelial cells, adding endothelial cells and then adding blood to the bottom chamber (Figure 1.5.2.1). This LOC model was used to test the effects of lipopolysaccharide (LPS), cytokines and infection on the COPD epithelium (Benam et al., 2016).

Figure 1.5.2.1- Schematic of the lung-on-a-chip model by Benam et. al: Basal epithelial cells are differentiated at air-liquid interface in the air channel and contain ciliated, goblet and basal cells on top of a semipermeable polyvinylidene fluoride (PVDF) membrane. Endothelial cells are cultured on the underside of the PDVF membrane in the chamber below where blood is pumped through to induce flow. The PDVF membrane can be stretched by inflation of side channels with air to induce stretch stimulus in the cells. The ability to strictly control independent parameters such as blood flow, cell types and soluble factors in the chip whilst obtaining a real time readout of results make this model very well suited to large high throughput screens for preclinical drug analysis. Furthermore, if multiple organ chips can be connected, systemic effects of drugs can be screened (Huh et al., 2010, Benam et al., 2016). Although lung on a chip models have been described as 3D models, the inherent cell culture nature of the chip is 2D as cells contained within them are grown as monolayers. In addition, the cells are still grown on stiff surfaces that do not replicate the cell-ECM interaction in vivo. Therefore, they may not be suitable as models of development and disease progression.

1.5.3 Organoid Models The terminology, organoids has been used since the early 1970s to describe an array of different models such as fibroblast-epithelial co-cultures, explants and resections with the term being utilised indiscriminately. With the advent of colon organoids at the start of the decade using the, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)+ system. Here, the progenitor cells were used to generate spheroids with lumens that generated the specific architecture of the differentiated colon lumen, together with small crypts that allow for the regeneration of the cell population (Clevers, 2016).

In this thesis the term organoid will be defined as:

“3D structures derived from stem/progenitor cells that self-organise to generate the cellular architecture of the native organ from which they were derived”.

Organoid models from several organs have been developed recently such as brain (Hattori, 2014), bone (Kale et al., 2000), colon (Sato et al., 2011), liver (Huch et al., 2015), lung (Danahay et al., 2015), mammary gland (Dontu et al., 2003), pancreas (Huch et al., 2013), prostate (Gao et al., 2014), small intestine (Sato et al., 2009) and stomach (Barker et al., 2010). Several different techniques have been employed and different growth factor-containing media used depending upon the specific tissue to generate organ-specific differentiation.

Primary HBE embedded in matrigel autonomously organise into spheroidal structures that form an inner differentiated luminal epithelium containing cilia (FOXJ1/α-Tubulin+) and goblet (MUC5AC+) cells. These organoids are derived from ITGA6+, P63+ and NGFR+ basal epithelial cells and have been termed tracheospheres or bronchospheres depending on the tissue from which they were derived (Figure 1.5.2.2) (Danahay et al., 2015). Other distal cell types such as club cells have not been observed. Recent work has sought to also create alveolar spheres with very limited success and currently ATI/ATII cell organisation into alveolar spheres do not replicate the structures observed in vivo (Barkauskas et al., 2013).

Lung organoid models have potential for studying development, disease and repair mechanisms since cellular differentiation is controlled by basal epithelial cells. For example, recent experiments by Danahay et al. have elucidated NOTCH2 as a mediator of goblet cell metaplasia as a downstream response to interleukin (IL-13) stimulation. Other studies have shown that treatment of mouse tracheospheres with FGF2, FGF9, FGF10 and ROCK inhibitors can have effects on cell size and morphology (Hegab et al., 2015) that could be applied to patient-derived bronchospheres.

Figure 1.5.2.2-Lung organoid: Integrin subunit α 6 (ITGA6)+, tumour protein p63 (P63)+ and nerve growth factor receptor (NGFR)+, basal epithelial cells aggregate and differentiate into Forkhead box J1 (FOXJ1)+/α-Tubulin+ ciliated cells and mucin 5AC (MUC5AC)+ goblet cells when grown in matrigel.

Several studies have investigated the susceptibility of lung organoids to infection by influenza virus. A study by Hui et. al compared the infection of lung organoids and bronchial explants with influenza virus. The study found that organoid infection was comparable to that of human explants and provided a physiological model for studying viral infections (Hui et al., 2018). These data were confirmed by Zhou et. al, (Zhou et al., 2018).

Currently, there is a great drive in the lung development field to generate organoid models from human pluripotent stem cells (hPSC). A landmark study by Chen et al. (Chen et al., 2017), showed that lung organoids could be differentiated into budding structures similar to those observed during lung development by step-by-step mimicking of the environmental conditions during definitive endoderm, anterior foregut endoderm and ventral anterior foregut endoderm morphogenesis using growth factors (Chen et al., 2017). The resulting lung bud organoid (LBO) recapitulated many features of the embryonic lung in the second trimester after ~170 days in culture. LBO showed markers for distal cell populations including ATI and ATII cells at the saccular tip and MUC5AC+ and SCGB3A2+secretory cells more proximally. A distal-to-proximal axis was also shown by the gradient of SOX9+ to SOX2+ expression. The model was used to examine the effects of respiratory syncytial virus (RSV) infection. Pulmonary fibrosis was also modelled by Crispr-Cas9 deletion of endothelial heat shock protein 1

(HSP1), which resulted in the accumulation of ECM around the LBO (Chen et al., 2017).

Unlike ALI cultures, there is no standardised methodology for bronchosphere culture as the system has only really taken hold in the last 5 years in the respiratory field. Published studies culture bronchospheres from 14-56 days using different ECM compositions including collagen only and matrigel ECM. Protocols also use media with different growth factors. As previously discussed in this chapter, differences in the local microenvironment can have profound effects on cell differentiation and by extension on organoid development. The length of time required for organoid culture using most protocols make the use of lung organoids unfeasible in medical diagnostics where quick readouts are required. In addition, the costs of culture are prohibitive for most laboratories.

However, these issues stem from the infancy of the technique and time may allow protocols to be optimised and standardised for use in different areas of the respiratory field.

1.5.4 Future Directions It is clear that respiratory research has now developed to the point where investment is needed in novel models that can answer key questions in human lung development, regeneration and repair. Whilst there is considerable debate about which model is better, it is important to consider that the model that is best for each specific research question should be used. Multiple models may need to be used to obtain an answer, for example a study concerning barrier function may utilize ALI, lung on a chip and mouse models whereas studies concerning differentiation of clonal populations of stem cells could use organoids and further validate results by implantation into mice. Novel organoid models, whilst utilising cells from the patient may not be able to inform on systemic effects of pharmacological agents, but may be informative on drug efficacy in humans, streamlining the number of pharmacological agents that pass to downstream LOC, animal models and clinical trials. Therefore, there is a potential role for the use of organoid models to reduce the use of animals in research and better direct translational and therapeutic research.

Currently, devices such as LOC have incorporated microenvironmental components into the culture that makes LOCs a powerful tool for studying a variety of cell-cell-environment interactions. Presently, no other culture system incorporates mechanical stimuli such as flow or stretch as stimuli during culture (Danahay et al., 2015, Chen et al., 2017). However, the LOC model is limited in its adaptability by the types of cell-cell interactions that can be studied as only immune, vasculature or epithelial cell interactions can be studied (Huh et al., 2010). Other parts of the respiratory system like bronchioles have ASM, fibroblast, ECM, basement membrane as well and play a major role in cell differentiation and development. LOC is therefore not sufficiently adaptable for this purpose without the connection of multiple LOCs which may not be feasible for everyday laboratory experimentation. The future promise of connecting multiple organ LOCs together, not only from different parts of the respiratory tract, but from different organs such as the liver and heart is an advantage that cannot be matched by organoid cultures (Ishida, 2018). Thus, LOCs could potentially be the best in vitro models for studying the systemic effects of disease using healthy subject or patient tissues. This will allow the reduction in the use of animal models in disease and particularly toxicological research.

Potentially, organoid culture may be more adaptable than LOC models as many different cell types can be added to the ECM scaffold in culture. The adaptability of the culture makes it a better model to study developmental processes and cell-cell signalling as cells can organically associate in culture to create their own junctions without the interference or separation by membranes (Huh et al., 2010, Danahay et al., 2015). As previously discussed, junctional signalling is important in development, differentiation and repair. One of the major drawbacks of 2D models of lung disease is their utilisation of unmodifiable materials such as plastic surfaces. Plastic surfaces are stiff and may affect cellular signalling and therefore the behaviour of progenitor cells.

An advantage of organoid models is that the matrix-cell interaction occurs in a 3D environment. As described previously, cells signal through the matrix by secreting signalling molecules that are bound to the matrix and which are then accessed by other cells (Section 1.2.2 and 1.2.3). Thus, information can be passed from cell to cell in a matrix concentration-dependent manner (Section 1.2.2 and 1.2.3). Organoid 65

models may be better able to be manipulated to study inter-cell communication via the matrix. Cells mechanically sense their environment through cell-cell-matrix interactions, aggregating to form tissue-specific morphologies (Section 1.2.2 and 1.2.3 and 1.3.0). The shape of an organ may be important to its function which may be masked by the 2D nature of LOC and other cultures. Therefore, matrix and surface stiffness and composition are important in apical-basal identity acquisition, lung tubule formation, cellular differentiation and repair. The imbalance in matrix composition that causes excess stiffness is seen in diseases such as IPF and COPD. As witnessed by MDCK models, 3D gel-based models reorganise the matrix around them and form a microenvironment that overcomes this mechanical variable (Muro et al., 2008).

Lung organoid models have started to be used in lung research, mainly in the field of developmental biology, although their efficacy in assessing disease is yet to be clarified. Organoid co-culture is under developed and investment in optimising experimental conditions for these kinds of assays are required before widespread use of complex organoid models can be undertaken. No organoid models for lung obstructive diseases such as asthma, COPD and IPF exist compared to other available lung models. Organoid models made from patient samples of lung obstructive diseases such as COPD, may reveal novel information about disease pathophysiology, development and progression in vitro and may be a novel platform for study and therapeutic discovery.

1.6 Hypothesis CHBE cells will form bronchospheres (CHBE-B) that are remodelled in comparison to bronchospheres formed from NHBE cells (NHBE-B) and that this will be associated with dysregulated expression of COPD- and development-associated genes. Furthermore, these differences can be mimicked by exposure of NHBE-B to cigarette smoke which is a major driver of COPD inflammation and airway/lung remodelling. Therefore, chronic treatment of NHBE cells with cigarette smoke condensate (CSC-HBE) during bronchosphere formation, will drive the bronchosphere phenotype towards a CHBE-B phenotype and away from an NHBE-B phenotype.

1.6.1 Aims • Establish and optimise a protocol for bronchosphere culture using NHBE cells and characterise the model. • Use an optimised protocol to grow bronchospheres from cigarette smoke treated NHBE (CSC-HBE) and from COPD HBE (CHBE) cells and characterise the models. • Compare and investigate similarities and differences between NHBE, CSC- HBE and CHBE bronchosphere gene expression profiles and on functional outputs. • Investigate the effect of co-culture with HASM and human lung fibroblasts (NHLF) from healthy subjects on bronchosphere formation.

2 Chapter 2

Methodology

2.1 Monolayer Cell Culture

2.1.1 HBE cell culture The reagents used for the culture of HBE cells are shown in Table 2.1.1. All cells were obtained from commercial sources or from the Airways Disease Biobank. Informed consent was obtained where necessary, full details are provided in the individual results chapters.

Table 2.1.1-Bronchial epithelium cell growth medium (BEGM): epidermal growth factor (EGF), Epinephrine, Hydrocortisone, Insulin, Transferrin and triiodothyronine were added to 500 ml BEBM. Concentration Volume Reagent Supplier (mg/ml) (ml) Lonza, Basel, Switzerland/ Bronchial Epithelium Basal - 500 Promocell, Medium (BEBM) Heidelberg, Germany EGF 0.5 0.5 Lonza/Promocell Epinephrine 0.5 0.5 Lonza/Promocell Hydrocortisone 0.5 0.5 Lonza/Promocell Insulin 0.005 0.5 Lonza/Promocell Retinoic Acid (RA) 0.5 0.5 Lonza/Promocell Transferrin 10 0.5 Lonza/Promocell Triiodothyronine 0.0067 0.5 Lonza/Promocell Trypsin/EDTA 0.04/0.03% 100 Lonza/Promocell Trypsin Neutralising Solution 0.05/0.01% 100 Lonza/Promocell (TNS) Trypsin Inhibitor/BSA

NHBE or CHBE cells were removed from liquid nitrogen and thawed by immersion of half the cryovial in a water bath at 37°C for 2 minutes. Cells were pipetted into a T150 flask containing 30 ml BEGM and allowed to adhere overnight in a 37°C humidified 5% CO2 incubator. Media was changed every 2 days thereafter until flasks became 90% confluent.

2.1.1.1 Passage of HBE cells BEGM was removed and cells were washed in 5 ml phosphate buffered saline (PBS) 3 times. Cells were then incubated with trypsin/Ethylenediaminetetraacetic acid

(EDTA) (0.04%/0.03%) for 3 minutes in a 37 °C humidified 5% CO2 incubator. The flask was gently tapped to remove adherent cells and 100 μl/cm2 of Trypsin

Neutralising Solution (TNS) was added to inactivate the trypsin. Cells were transferred to a 50 ml falcon tube and pelleted at 220 x g for 5 minutes. Cells were then resuspended in fresh 5ml BEGM and were seeded into a new T150 flask at 6,666 cells/cm2 in 30ml.

Cells were not passaged beyond passage 2 using this culture method as it resulted in poor bronchosphere generation. Cells were frozen at passage 1 at 5 x106 cells/ml in 1 ml cryoSFM media in cryovials in a Mr. Frosty at -80 °C overnight before being transferred to a liquid nitrogen tank at -195 °C for long-term storage.

2.1.2 Stromal cell culture All media required for stromal cell culture are given in Table 2.1.2. Healthy lung fibroblast (NHLF) or healthy airway smooth muscle (NHASM) were removed from liquid nitrogen and thawed by immersion of half the cryovial in a water bath at 37°C for 2 minutes. Cells were pipetted into a T150 flask containing 20 ml complete Dulbecco’s modified eagle Eagle’s medium (DMEM) and allowed to adhere overnight in a 37 °C humidified 5% CO2 incubator. Media was changed every 2 days thereafter until flasks became 90% confluent.

2.1.2.1 Passaging of NHLF and NHASM cells Complete DMEM was removed and cells were washed in 5 ml PBS 3 times. Cells were then incubated with trypsin 0.5 g/ml for 3 minutes in a 37 °C humidified 5% CO2 incubator. The flask was gently tapped to remove adherent cells and 100 μl/cm2 of complete DMEM was added to inactivate the trypsin. Cells were transferred to a 50 ml falcon tube and pelleted at 220 x g. Cells were suspended in fresh 5 ml DMEM and seeded into a fresh T150 flask at 6,666 cells/cm2 in 20 ml.

Table 2.1.2-Complete DMEM: 500 ml DMEM contained 10% FCS, 2 mM L-Glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

Reagent Volume (ml) Supplier Dulbecco’s Modified Eagle’s Sigma-Aldrich, St. 500 Medium (DMEM) Louis, USA Foetal Calf Serum (FCS) 50 Sigma-Aldrich L-Glutamine (200 mM) 5 Sigma-Aldrich Penicillin/Streptomycin 5 Sigma-Aldrich (0.1 mg/ml)

2.1.3 Feeder Layer Culture All reagents used for 3T3 feeder cell culture are provided in Table 2.1.3.

Table 2.1.3-3T3 medium: 500 ml DMEM contained 10% BS, 2 mM L-Glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Reagent Volume (ml) Supplier Dulbecco’s Modified Eagle’s Medium 500 Sigma-Aldrich (DMEM) Bovine Serum (BS) 50 Sigma-Aldrich L-Glutamine 5 Sigma-Aldrich Penecillin/Streptomycin 5 Sigma-Aldrich

3T3-J2 mouse embryonic fibroblasts were expanded in 3T3 media until 70% confluent. 3T3 cells were cultured at sub confluence to ensure that the cell phenotype was retained (Butler et al., 2016).

2.1.4 HBE-3T3 cell co-culture All reagents used for HBE-3T3 cell co-culture are provided in Table 2.1.4.

Table 2.1.4-Co-culture medium (CCM): 3T3 complete DMEM (10% FCS, 2 mM L-Glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin), 0.4 mg/ml Cholera Toxin, EGF, Hydrocortisone, Insulin and 0.005 mM Y-27632 as described in (Butler et al., 2016). Reagent Volume (ml) Supplier Dulbecco’s Modified Eagle’s 500 Sigma-Aldrich Medium (DMEM) Ham’s F-12 nutrient mixture 500 Gibco L-Glutamine 5ml Sigma-Aldrich Cholera Toxin 0.0043 Sigma-Aldrich EGF 0.5 Lonza/Promocell Hydrocortisone 0.5 Lonza/Promocell Insulin 0.5 Lonza/Promocell Mitomycin 0.5 Sigma-Aldrich Penecillin/Streptomycin 5 Sigma-Aldrich Y-27632 0.5 Sigma-Aldrich

HBE-3T3 cell co-culture feeder layers at 70% confluence were treated with mitomycin C (0.4 mg/ml) in 15 ml 3T3 media for 2 hours before being seeded into fresh T75 flasks at 2x105 cells/cm2 overnight. 3T3 media containing mitomycin was removed, the culture was washed 3 times with 5 ml PBS and 1x106 HBE cells were seeded in 15 ml co-culture medium (CCM). Media was changed every 2 days thereafter. 71

After 3 days colonies of epithelial cells were observed. As colonies expand 3T3 cells will lift off. Epithelial cells were passaged when they reached 90% confluency.

2.1.4.1 HBE-3T3 co-culture passaging Fresh 3T3 feeder layers were prepared the day before passaging as described above. 3T3 fibroblasts were differentially trypsinised by incubation with trypsin/EDTA (0.04%/0.03%) for 2 minutes at room temperature. Cells were washed 3 times with 5 ml PBS before being incubated with trypsin/EDTA for a further 3 minutes before the addition of TNS. Cells were pelleted at 220 x g and resuspended in fresh CCM. Cells were seeded onto feeder layers at 6,666 cell/cm2 in 15 ml.

For HBE expansion, cells were always passaged on mitomycin C-treated 3T3 feeder layers. Theoretically, HBE cells can be expanded using this culture indefinitely. However, cells were not expanded beyond passage 14 (Butler et al., 2016). HBE were not directly used from this culture, but were further grown in BEGM media and split once to ensure that 3T3 fibroblast cross contamination did not occur as 3T3 fibroblast are unable to survive in BEGM (Hynds et al., 2016). The cell culture method was adapted from (Butler et al., 2016). Further details are available from this reference

2.2 Bronchosphere Culture All reagents used for bronchosphere culture are provided in Table 2.2.

Table 2.2-Differentiation medium: 500 ml 1:1 BEBM:DMEM contained EGF, Epinephrine, Hydrocortisone, Insulin and Transferrin. RA was added at time of use for a final concentration of 100 nM. Concentration Reagent Volume (ml) Supplier (mg/ml) Bronchial Epithelium Basal - 500 Lonza/Promocell Medium (BEBM) Gibco, Dulbecco’s Modified - 500 Loughborough, Eagle’s Medium (DMEM) UK EGF 0.5 0.5 Lonza/Promocell Epinephrine 0.5 0.5 Lonza/Promocell Hydrocortisone 0.5 0.5 Lonza/Promocell Insulin 0.005 0.5 Lonza/Promocell All-Trans Retinoic Acid 0.5 0.5 Sigma-Aldrich (RA) Corning, New Matrigel - 10 York, USA Labcyte, San Microclime Plate Lids - - Jose, USA Transferrin 10 0.5 Lonza/Promocell Trypsin/EDTA 0.04/0.03% 100 Lonza/Promocell Trypsin Neutralising 0.05/0.01% 100 Lonza/Promocell Solution (TNS) Ultra-Low attachment 96 - - Corning well plates

Plates were coated with 25% matrigel in differentiation media supplemented with

100 nM retinoic acid (RA) overnight in a 37 °C humidified 5% CO2 incubator. HBE cells were cultured as monolayers as described above (2.1.1.1), trypsinised and pelleted by centrifugation at 220 x g for 5minutes. HBE were resuspended at 38,462 cells/ml in 5% matrigel in differentiation media supplemented with 100 nM RA and were pipetted gently into matrigel coated wells (Figure 2.1). Microclime plate lids were filled with 10 ml cell culture grade H2O supplemented with penicillin-streptomycin and amphotericin and were placed on the plates. Plates were pulse centrifuged at 500 rpm and incubated in a 37 °C humidified 5% CO2 incubator. Cells were fed on day 2 and every 6 days thereafter with 70 μl of 5% matrigel in differentiation media supplemented with 100 nM RA for 20 days. 73

Figure 2.1-Schematic of bronchosphere culture- (A) Matrigel was mixed with differentiation media containing 1:1 basal epithelial growth medium without triiodothyronine and supplemented with 100 nM retinoic acid with Dulbecco’s Modified Eagles Medium to make 25% matrigel which was pipetted into wells at 10 μl and incubated for 20 minutes at 37 °C, (B) epithelial cells in were seeded onto 25% matrigel layer in 5% matrigel and were fed on day 2, 8 and 14 of culture with 5% matrigel until beating cilia were observed.

Care was taken not to disturb cells by keeping the incubator door closed until the day of feeding. Bronchosphere development is highly affected by changes in humidity and heat.

2.3 Bronchotubule Culture NHBE cells were expanded using the 3T3-J2 feeder layer method (Section 2.1.4 above) and then were cultured as monolayers (Section 2.1.1.0 above).

2.3.1 Matrigel Overlay Culture NHLF cells were cultured as described above (Section 2.1.2.0) before being removed from the flask by trypsinisation. NHLF were seeded onto 96 well plates at either 25,000, 75,000, 225,000 or 675,000 cells/ml in 0.1 ml differentiation media and were allowed to adhere overnight. The next day media was removed, and cells were coated with 0.05 ml 25% matrigel in differentiation media supplemented with 100 nM

RA. Matrigel was allowed to set for 2 h at 37°C humidified 5% CO2 incubator.

Figure 2.2-Schematic of Matrigel overlay culture. (A) Matrigel was mixed with differentiation media containing 1:1 basal epithelial growth medium without triiodothyronine and supplemented with 100 nM retinoic acid with Dulbecco’s Modified Eagles Medium containing NHLF at either 25,000, 75,000, 225,000 or 675,000 cells/ml to make 25% matrigel which was pipetted into wells and incubated for overnight at 37°C, (B) 25% matrigel layer supplemented with 100 nM retinoic acid was added and incubated at 37°C for 2 hours (C) epithelial cells in 5% matrigel were added at 50,000 cells/ml.

NHBE cells were cultured as monolayers as described above (Section 2.1.1.0). NHBE cells were seeded onto wells at 50,000 cells/ml in 0.05 ml 5% matrigel in differentiation media supplemented with 100 nM RA (Figure 2.2). The cells were incubated until bronchotubules collapsed and formed spheres (4-6 days depending on patient).

2.3.2 Matrigel Triple Culture 96 well plates were coated with 50 μl 25% matrigel in differentiation media supplemented with 100 nM RA. NHBE, NHLF and NHASM were grown in monolayers as described above (Section 2.1.2.0). NHBE, NHLF and NHASM cells were trypsinised from plates and resuspended in media before being mixed together at 25,000:675,000:675,000, 75,000:675,000:675,000, 225,000:675,000:675,000 or 675,000:675,000:675,000 cells/ml (NHBE:NHLF:NHASM respectively) in 100 μl 25% matrigel in differentiation media supplemented with 100 nM RA (Figure 2.3). Cells were co-cultured until tubules collapsed into spheres (6-8 days depending on patient).

Figure 2.3-Schematic of Matrigel triple culture. (A) Matrigel was mixed with differentiation media containing 1:1 basal epithelial growth medium without triiodothyronine and supplemented with 100 nM retinoic acid with Dulbecco’s Modified Eagles Medium containing 25,000:675,000:675,000, 75,000:675,000:675,000, 225,000:675,000:675,000 or 675,000:675,000:675,000 cells/ml (NHBE:NHLF:NHASM respectively) to make 25% matrigel which was pipetted into wells and incubated for overnight at 37°C, (B) culture was fed with 5% matrigel layer supplemented with 100 nM retinoic acid. 2.3.3 Agrigel* (agarose-matrigel mix) Culture Transwells were coated with 0.5%/25% or 0.7%/25% agrigel* and allowed to cool at room temperature for 5 minutes. Matrigel transwells were coated with 25% matrigel and were allowed to gel in a 37°C incubator 5% CO2 humidified incubator.

NHBE, NHLF and NHASM cells were trypsinised and centrifuged at 220 x g and cells were mixed in 50 ml falcon tubes at NHBE 45,000 cells/well and NHLF and NHASM at 67,500 cells/well. Falcon tubes were once again centrifuged at 220 x g and cells were resuspended in 25% matrigel in differentiation media. Low gelling agarose was melted using a microwave until no particulates could be observed and allowed to cool for 30 seconds before being mixed** with the re-suspended cells in matrigel and pipetted into the transwells. The gel was allowed to solidify at room temperature for 5 minutes and differentiation medium was added to the basal wells (Figure 2.4). The plates were incubated in a cell culture incubator for 20 days. Cells were fed with fresh media every 2 days.

Figure 2.4-Schematic of Matrigel overlay culture. (A) Culture was fed basally with 5% matrigel containing 1:1 basal epithelial growth medium without triiodothyronine and supplemented with 100 nM retinoic acid with Dulbecco’s Modified Eagles Medium, (B) empty matrigel (final concentration 25%) or matrigel and agarose (final concentration 0.5%) was mixed with differentiation media containing 1:1 basal epithelial growth medium without triiodothyronine and supplemented with 100 nM retinoic acid with Dulbecco’s Modified Eagles Medium (25/0.5% Agrigel) was pipetted into transwells and incubated at room temperature for 5 minutes, (C) 25/0.5% Agrigel containing either 25,000:675,000:675,000, 75,000:675,000:675,000, 225,000:675,000:675,000 or 675,000:675,000:675,000 cells/ml (NHBE:NHLF:NHASM respectively) was incubated at 37°C (D) culture was fed apically with 5% matrigel layer supplemented with 100 nM retinoic acid.

*Agrigel (Agarose/matrigel) was formed by mixing matrigel and agarose in differentiation media to a final concentration of 0.5% or 0.7% agarose and 25% matrigel. Matrigel was stored on ice until used. Matrigel was mixed with media prior to the addition of agarose and used when the solution turned pink. **Pipette tips were warmed and cut prior to mixing of agarose and matrigel. Agarose was used at 45 °C.

2.4 Generation of Cigarette Smoke Condensate (CSC) The materials and equipment used to produce CSC are describe in Table 2.4.1.

Table 2.4.1-CSC reagents Material Supplier

Smoking Robot VC10 (VC10) Vitrocell, Waldkirch, Germany

92mm Cambridge Filter-Pad (CFP) Vitrocell

University of Kentucky, Kentucky, 3R4F Cigarettes USA Sterile Filtered DMSO Sigma-Aldrich

50ml Falcon conical tube VWR, Randor, USA

The dry weight (W0) of 92 mm Cambridge Filter-Pad (CFP) were measured (to the nearest 0.1 mg) and 60 3R4F research grade cigarettes were smoked using the VC10 smoking robot. The resultant cigarette smoke (CS) was passed through the CFP and the total particulate matter (TPM) was collected. The CFP was removed and the wet weight (W1) was measured (to the nearest 0.1 mg) and the weight of TPM was determined according to the formula below:

TPM (mg/cigarette) = (W1- W0)/Q Where Q is the number of cigarettes smoked through the CFP

The CFP was folded twice with the TPM side facing inwards and transferred to a 50 ml conical flask. 9ml of dimethyl sulfoxide (DMSO) was added and the flask was placed on a shaker for 20 minutes followed by 20 minutes of sonication. The recovered solvent containing solubilized TPM is defined as CSC which was filter sterilized through a 0.22 M filter. 125 mg/ml aliquots of CSC were stored in single use amber vials at -80 °C.

The bioactivity of CSC was ascertained at Novartis. HBECs grown at air-liquid interface (ALI culture) were exposed on the basolateral side with 1, 10 and 100 g/ml of CSC for 72 h and the mRNA expression of the cytochrome P450 enzymes CYP1A1 and CYP1B1 measured using reverse transcription (RT)-quantitative polymerase

chain reaction (QPCR). These monooxygenases are induced by polycyclic aromatic hydrocarbons found in cigarette smoke.

CSC loses bio-activity upon extended storage and therefore fresh CSC aliquots were produced every 3 months. The batch to batch variability of CSC activity was tested periodically on HBEC ALIs to maintain the consistency of CSC.

2.5 Cell Viability Assay The reagents required for the cell viability assay are provided in Table 2.5.

Table 2.5- MTT reagents Reagent Supplier

3-(4, 5-Dimethylthiazol-2-yl)-2, 5- Sigma-Aldrich phenyltetrazoliumbromide (MTT) DMSO Sigma-Aldrich Fluostar Optima Fluorimeter BMG LabTech, Aylesbury, UK PBS Sigma-Aldrich

The 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-phenyltetrazoliumbromide (MTT) assay was used to determine cell viability. The yellow MTT dye is reduced by active mitochondria to purple formazan crystals that are dissolved with DMSO. The number of viable cells is proportional to the absorbance reading. Cells cultured in 96 well plates were centrifuged at 220 x g, the supernatant was discarded, and the cells were incubated for 2 hours with 100 μl of 1mg/ml MTT solution. The generation of the purple formazan product was confirmed via light microscopy. Formazan crystals were dissolved in 100 μl DMSO for 5 minutes with shaking. The absorbance was measured at 550 nm using a spectrophotometer. Cell viability was calculated as a percentage of optical density (OD) relative to untreated control cells.

2.6 Cilia Beat Frequency (CBF) Cilia beating in 4 bronchospheres was imaged at 100x using a Leica DMLB upright microscope with a trouble shooter high speed camera at 500 frames/second. The video was played back at 60 frames/second and the number of frames needed to

complete 10 cycles of cilia beating were counted. The conversion to CBF used the following formula:

CBF (Hz) = 500 frames/sec / (number of frames/number of beats)

3 areas containing cilia were counted in each of the 4 bronchospheres. Counts for each bronchosphere was averaged to give the CBF for that bronchosphere. The mean CBF of the patient was calculated by averaging the means for the 4 bronchospheres.

2.7 Bronchosphere RNA Extraction

2.7.1 Bronchosphere Isolation, lysis and homogenisation The reagents required for the RNA extraction are provided in Table 2.7.1.

Table 2.7.1-Isolation, lysis and homogenisation reagents Reagent Supplier β-mercaptoethanol Sigma-Aldrich Cell Recovery Solution Corning Percyllys, Montigny-le-Bretonneux, Percellys Tubes France QIAshredders™ homogenisers Qiagen RNase-free water Sigma-Aldrich

Prior to RNA extraction from bronchospheres, the 5% matrigel was slowly aspirated down to 50 μl being careful not to remove the bronchospheres. 0.06 ml/mm of corning cell recovery solution (CCR) was added to each well and incubated for 30 minutes at room temperature to release bronchospheres from the matrigel. Post aspiration, bronchospheres were transferred to a 6 well plate and centrifuged at 220 x g for 5 minutes. CCR solution was aspirated and replaced with RLT buffer (Qiagen) with 1% β-mercaptoethanol (β-MEM) to lyse bronchospheres and inactivate RNases. Bronchospheres were bead homogenised by shaking at 6500 rpm with percellys beads and centrifuging sample through Qiashredder column at 12,000 x g for 2 minutes.

2.7.2 RNA Extraction Total RNA was isolated from Bronchospheres using the RNeasy Mini Kit; all centrifugation was performed at room temperature and all buffers used were from RNeasy Mini Kit (Table 2.7.2). 70% ethanol was mixed 1:1 with sample lysate and centrifuged through RNeasy MinElute spin columns at 12,000 x g for 15 seconds to enable RNA binding to columns and the flow-through was discarded. Spin columns were washed with buffer RW1 by centrifugation 12,000 x g for 15 seconds. Any residual genomic DNA was removed by incubation with DNase for 15 minutes at room temperature.

Table 2.7.2- RNA extraction reagents Reagent Supplier RNeasy Micro™ Kit Qiagen RNase-free water Sigma-Aldrich

Columns were then washed with buffer RW2 and 80% ethanol by centrifugation 12,000 x g for 15 seconds and 2 minutes respectively. The columns were placed in fresh collection tubes with their lids open and were centrifuged at 12,000 x g for a further 5 minutes. Columns were placed in fresh collection tubes once more and the total RNA was eluted from the spin column in 14 μl RNase-free water by centrifugation for 1 minute at 12,000 x g. The quantity and purity of the isolated total RNA was measure using a tape station or a Nanodrop™ Lite (NanoDrop Technologies, Wilmington, USA). The total RNA concentration was given as ng/µl and the purity was determined by the A260 nm: A280 nm absorbance ratio. mRNA gave a ratio of between 1.8-2 relative units. Total RNA was stored at -80°C. Bronchospheres from wells treated in the same manner were pooled to generate enough RNA during the extraction step.

2.7.3 Reverse transcription 0.5 μg of total RNA was resuspended in 10 μl of RNase free water and was denatured at 70 °C for 5 minutes. Master mix containing components from Table 2.7.3 was added to the RNA and samples were incubated 42°C for 1h followed by an enzyme inactivation step at 90°C for 4 minutes to synthesise single stranded cDNA. Samples were diluted with 40 μl of DEPC-treated water after the reaction.

Table 2.7.3-Reverse polymerase chain reaction components

Reagent Volume (μl)/ well Final Concentration 5x AMV buffer 4 1x dNTP 2 1 mM Random primers 1 1 μg RNasin 1 40 U AMV RTase 1 10 U

H2O-DEPC 1 N/A

2.8 Immunofluorescence (IF) Staining The reagents and antibodies (primary and secondary) used for IF staining is indicated in Table 2.8.

Table 2.8- Immunofluorescence reagents Reagent Supplier Bovine Serum Albumin (BSA) Sigma-Aldrich Foetal Calf Serum (FCS) Sigma-Aldrich Phosphate Buffered Saline (PBS) Sigma-Aldrich Triton X-100 Sigma-Aldrich Tween-20 Sigma-Aldrich

Final Primary Antibody/Stain Animal/Epitope Supplier Concentration Acetylated α-Tubulin Mouse, Ig2 1/400 Sigma-Aldrich Abcam, E-Cadherin Mouse, Ig1 1/400 Cambridge, UK ITGA6 Mouse, Ig1 1/400 Abcam Muc5AC clone Mouse, Ig1 1/400 Sigma Santa Cruz, P63 Rabbit, Igg 1/400 Santa Cruz, USA

Final Secondary Antibody/Stain Animal/Epitope Supplier Concentration Thermo Fisher Scientific, Alexa Fluor 488 Mouse, Ig1 1/400 Loughborough, UK Thermo Fisher Alexa Fluor 568 Mouse, Ig2 1/400 Scientific Thermo Fisher Alexa Fluor 633 Rabbit, Igg 1/400 Scientific Thermo Fisher DAPI N/a 1/5000 Scientific

Monolayer cells/bronchospheres/bronchotubules were permeabilized and blocked in IF blocking buffer (IFB, 0.2% Triton X-100, 0.1% BSA, 0.05% Tween 20 in PBS, 10% FCS) for 1 h before incubation with primary antibodies and DAPI by shaking overnight at 4°C in IFB buffer. Samples were then washed with IF buffer (0.2% Triton

X-100, 0.1% BSA, 0.05% Tween 20 in PBS) for 5 minutes four times, IF buffer was discarded and samples incubated with secondary antibodies for 2 h at room temperature. Samples were then washed for 5 minutes 4 times in IF buffer and twice with PBS.

Images were visualized using Leica DMI6000 and analysed using imageJ version 1.52e (Wayne Rassband).

2.9 Maxiprep Plasmid Purification and Plasmid Generation The reagents used to grow, and isolate plasmids are provided in Table 2.9.1.

Table 2.9.1- Plasmid reagents Materials Supplier Yellow Fluorescence Protein (YFP) Plasmid Adgene, Cambridge, USA transfected bacteria mCherry Plasmid transfected bacteria Adgene pMD.2G Gifted from Crick Institute psPAX2 Gifted from Crick Institute Lauria-Bertani Broth Invitrogen, Carlsband, USA Ampicillin Invitrogen Qiagen Maxiprep Kit (all buffers used were Qiagen from this kit)

Yellow fluorescence protein (YFP), mCherry, pMD.2G or psPAX2 bacteria were streaked onto 100 μg/ml ampicillin supplemented agar plates and incubated overnight at 37°C. The plasmid maps for YFP an mCherry vectors are shown in Figure 2.9. Individual colonies were suspended in 5 ml of Lauria-Bertani broth (LB) broth supplemented with 100 μg/ml ampicillin and incubated for 4 hours at 37 °C with agitation. The culture was transferred to 245 ml LB broth supplemented with 100 μg/ml ampicillin and incubated overnight with agitation at 37°C before being centrifuged at 6,000 x g for 15 minutes at 4°C.

Figure 2.9-Schematic of yellow fluorescence protein (YFP) and mCherry plasmids: Left YFP cloned into a puro destiny plasmid pLEX_970_puro_DEST_YFP was a gift from William Hahn, Dana Faber Cancer institute (Addgene plasmid # 45295 and right mCherry cloned into plv plasmid pLV-mCherry was a gift from Pantelis Tsoulfas, University of Miami (Addgene plasmid # 36084).

YFP or mCherry bacterial pellets were re-suspended in 10ml of maxiprep buffer P1 supplemented with 100 μg/ml RNase and was mixed with 10ml maxiprep buffer P2 and incubated for 5 minutes. 10 ml pre-chilled maxiprep buffer P3 was added and the lysate was incubated on ice for 20 minutes before centrifugation for 30 minutes at 20,000 x g at 4°C. The supernatant was centrifuged under the same conditions for a further 15 minutes in a fresh tube. A QIAGEN-tip 500 gravity column was equilibrated by passing 10 ml of QBT Buffer through the column prior to passing the lysate through the column via gravity flow. The columns were washed with 30 ml of Buffer QC twice, and plasmid DNA was eluted with 15 ml Buffer QF into a fresh centrifuge tube. 10.5 ml of isopropanol was added to precipitate the DNA prior to centrifugation at 15,000 x g for 30 minutes at 4 °C.

The DNA pellet was washed with 70% ethanol and centrifuged for 15 minutes at 15,000 x g. The supernatant was decanted without disturbing the pellet and the tube was air dried for 10 minutes. The pellet was re-suspended in 300 μl of RNase and

DNase free H2O and quantified using a NanoDrop ND-1000 Spectrophotometer.

2.9.1 Lentiviral Generation The reagents used to generate lentiviral vectors are provided in Table 2.9.2.

Table 2.9.2- Lentiviral reagents Materials Supplier HEK293FT cells Gifted from Crick institute Heat inactivated FBS Invitrogen DMEM Invitrogen OPTI-MEM Invitrogen 6 well cell culture plate 9.5 cm2/well Corning Lipofectamine 2000 Invitrogen 0.45 μm polyethersulfone filter Sigma-Aldrich (Milex) Lenti-X™ Concentrator kit Takara Bioscience, Kasatu Japan Lenti-X™ qRT-PCR Titration Kit Takara Bioscience

1 x106 HEK293FT cells were plated into a 6 well cell culture plate in DMEM supplemented with 10% FBS and allowed to adhere overnight in a 37 °C humidified

5% CO2 incubator to generate either YFP or mCherry viral particles. Transfection components were prepared in separate tubes. The first tube contained 10 μg of YFP or mCherry, 7 μg of psPax2 and 3μg of pMD.2G (viral RNA plasmid envelope) in 0.5 ml OPTI-MEM and the second tube contained 0.5 ml OPTI- MEM and 30 μl lipofectamine 2000. The contents of both tubes were combined, vortexed and incubated at room temperature for 20 minutes to allow complex formation. The tubes were mixed and added to fresh DMEM to make a final volume of 4.5 ml. 1.5 ml of new DMEM solution was used to replace the cell media and cells were cultured in a 37 °C humidified 5% CO2 incubator. After 18 hours, the medium was replaced with fresh DMEM containing 10% FBS and the cells were left for a further 72 h for viral production. The viral supernatant was collected and filtered through a 0.45 μm polyethersulfone low protein binding filter and centrifuged at 1600 × g at 4°C for 10 minutes to remove cell debris. The viral supernatant was mixed at a ratio of 3:1 with Lenti-X concentrator solution, incubated for 1 h at 4°C and centrifuged at 1,500 x g for 45 minutes to concentrate the supernatant. The resultant viral pellet was resuspended 1:50 with native DMEM and aliquoted for storage at -80 °C. A Lenti-X™ qRT-PCR Titration Kit was used to calculate viral multiplicity of infection (MOI) of NHLF and NHASM. NucleoSpin RNA Virus Kit from Lenti-X™ 86

qRT-PCR Titration Kit was used to lyse, extract and purify viral RNA from an aliquot of viral supernatant. Samples at 12.5 μl were incubated with 6.5 μl DNase I, for 30 minutes during extraction to remove any residual DNA at 37 °C then at 70 °C for 5 minutes. Control and viral YFP and mCherry RNA were serially diluted 10-fold in dilution buffer and were transferred to a PCR plate in duplicate where the enzyme mix was added. A non-template control was included, and the plate was centrifuged at 1200 x g (4°C) for 1 minute to ensure the removal of bubbles and that the solution was aggregated at the bottom of the well. Samples were amplified by PCR using the following cycles:

RT Reaction- 42°C 5 min followed by 95°C 10 seconds qPCR x 40 Cycles- 95°C 5 sec followed by 60°C 30 seconds Dissociation Curve- 95°C 15 seconds followed by 60°C 30 seconds which was followed by (60°C–95°C)

All reagents for PCR reactions are provided in Table 2.9.3.

The viral RNA quantity was calculated from the Ct values obtained by measuring the copy number of the gene of interest against the standard curve formed using viral

RNA control (average copy number vs. average Ct (log scale)). The copy number was calculated using the equation below for each dilution.

(copies N calculated from Cts)(1000 ml)(50 μl elution) 퐶표푝푖푒푠/푚푙 = (150 μl sample)(2 μl added to each well)

NHLF or NHASM were plated at 3,000 cells/well of a 96 well plate in duplicate and 1:2 fold dilutions of the viral supernatant was added to each well. YFP and mCherry were visualised using a Zeiss Axiovert 200 M confocal microscope and the volume of supernatant required to transfect cells was determined. An MOI of 6 was calculated as the maximal transfection for both cell types.

Table 2.9.3. Components, concentrations and volume of viral titre real time polymerase chain reaction (PCR) master mix. Table indicating the components which made up the reaction mix used for the viral titre calculation.

Reagent Volume (μl)/ well Supplier RNase-Free Water 8.0 Takara Bioscience Quant-X Buffer (2X) 12.5 Takara Bioscience Lenti-X Forward Primer 0.5 Takara Bioscience (10 μM) Lenti-X Reverse Primer 0.5 Takara Bioscience (10 μM) Reference Dye LSR 0.5 Takara Bioscience Quant-X Enzyme 0.5 Takara Bioscience RT Enzyme Mix 0.5 Takara Bioscience RNA 2 Takara Bioscience

The Lenti-X concentrator kit allows concentration of the virus without the need for ultra-centrifugation. Viral particles were aliquoted into single use aliquots as repeat freeze thawing reduces viral transfection efficiency. The Lenti-X™ qRT-PCR Titration Kit utilises PCR to generate a standard viral RNA control that can then be used to calculate the copy number of the virus-containing sample. Protocols were adapted from the Lenti-X concentrator and QPCR kits.

2.10 Rheometry of Agarose, Matrigel and Agrigel 50 µl of either 25% matrigel, Agarose (0.3, 0.5, 0.7 or 1.0%) or Agrigel (0.3/25, 0.5/25, 0.7/25 or 1.0/25%) solution was prepared as described in Section 2.3.3 above and instantly poured into a 10 mm diameter Teflon mould. Gels were allowed to dry at

37°C/5% CO2 for 20 minutes before incubation for 24 h in differentiation media containing all supplements. A rheometer discovery HR1 (TA Instruments, New Castle, USA) was used to measure the gels. At the time of measurement, the base plate was heated to 37°C and gels were removed from the mould and compressed with a 10 mm diameter compressive plate. An angular frequency ranging from 0.1 to 25 rad/s was used to acquire the storage modulus (G’) and the loss modulus (G”) of the gel solutions using TRIOS software (TA Instruments).

2.11 RNA-Seq Bioinformatics RNA from bronchospheres was analysed for quality using a tape station and Poly-Art RNA-Seq was performed by Novartis. Data was analysed by Dr Vahid Elyasigomari at the Data Science Institute at Imperial College London. Briefly, fastqc was run to check data quality before being aligned and counted using STAR. NHBE and CHBE cells were compared at day 0 along with the time course of bronchosphere development in NHBE-B and CHBE-B. Thus, NHBE-B and CHBE-B at day 8 and 20 were compared to their respective day 0 data using the OmicSoft Array Studio (v 7.0) (OmicSoft Corporation, NC, USA) using a general linear model. Subsequent pathway analysis was performed in EnRichR (Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013;128(14)). The effect of exposure to CSC was analysed in NHBEs at 2 h and in CSC-NHBE-B at days 8 and 20 compared to NHBE-B data. Further details of the analysis performed are provided in Chapter 4.

2.12 Statistical Analysis Graphpad Prism version 5.0 (San Diego, USA) was used to perform statistical analyses. Data were represented as mean ± SEM. Kruskal-Wallis test with Dunn’s post-test were used for non-parametric and analysis of variance (ANOVA) test with Bonferroni correction was for parametric data analysis.

3 Chapter 3

Bronchosphere Model Development and Characterisation

3.0 Introduction There are currently very few methods in the literature that describe lung organoid culture. To develop a protocol for lung organoid culture existing published methodologies were reviewed and experimental parameters that best replicated the lung environment were chosen as explained below. Methods for organoid culture varied in approach however, all methodologies are reliant upon early passage (p) of mouse/human basal epithelial cells (p1-3), a type of ECM scaffold and a differentiation medium (see methods) (Barkauskas et al., 2013, Tata et al., 2013, Chen et al., 2012, Danahay et al., 2015).

Human basal epithelial cells were used to create bronchospheres owing to the differences between the mouse and human stem cell population in the lung as previously discussed (Introduction, section 1.3.0). These cells were harvested from the bronchus and not the trachea since although COPD is observed in the more distal airways (Introduction, section 1.1.0), small airway epithelial cells were more difficult to obtain.

Different types of ECM scaffolds have been used for organoid culture such as collagen I- and Matrigel-based scaffolds (Barkauskas et al., 2013, Tata et al., 2013, Chen et al., 2012, Danahay et al., 2015, DiMarco et al., 2015, Islam et al., 2013). As previously stated, (Introduction, section 1.2.3), the BM in vivo contains very low levels of collagen I and is composed primarily of laminin and collagen IV (Harunaga et al., 2014). Therefore, organoid culture methods using only one type of ECM such as a collagen I scaffold are less representative of the in vivo environment of the basal layer of the lung.

Matrigel ECM scaffold is derived from an Engelbreth-Holm-Swarm (EHS) tumour, which is a benign tumour in mice mainly composed of BM. It contains approximately 60% laminin, 30% collagen IV, and 8% entactin as well as HSPG, nidogen and proteoglycan ECM proteins and is similar in composition to healthy human BM (Hughes et al., 2010, Sannes and Wang, 1997). Since the type of ECM proteins present can affect the morphology and differentiation of progenitor cells, matrigel was chosen over other scaffolds for organoid culture.

Recent publications have shown the use of decellularized human lung ECM to grow organoids. However, this technology was not available at the beginning of the project and could not be considered (Hedstrom et al., 2018).

Various protocols use different percentages of matrigel for organoid culture. Higher percentages of matrigel (>50%) result in organoids derived from a single cell, which makes this type of culture very powerful in terms of clonogenic studies (Rock et al., 2009). Cells seeded onto ~25% matrigel scaffold form organoids through autonomous aggregation into a heterogenous population of spheroids that differentiate into organoids (Barkauskas et al., 2013, Tata et al., 2013, Chen et al., 2012, Danahay et al., 2015). This project aims to use organoid models to study COPD, which requires a heterogenous population of epithelial cells to be present to mimic that seen after CS exposure. Therefore, 25% matrigel will be used to ensure bronchospheres are generated from an aggregate of heterogenous cells. Matrigel when first extracted contains mouse derived growth factors that are bound to the ECM. To ensure that basal cells are only exposed to human growth factors that are essential for lung development, growth factor reduced (GFR) matrigel was used in bronchosphere culture (Hughes et al., 2010).

Finally, differentiation medium used in ALI culture was used in all methodologies and was made from a 1:1 mix of high glucose DMEM-F12/growth media mix. All possible growth factors were added to the growth medium except for triiodothyronine, which has been shown to be inhibitory to goblet cell formation in ALI cultures, and RA which was added exogenously at the time of feeding. Some protocols add Y-27632, a ROCK inhibitor, in the first 48hrs of culture to ensure cell survival. Since this differentiation medium has been successfully used in ALI culture and has been well characterised it will be used to seed cells for lung organoid culture (Barkauskas et al., 2013, Tata et al., 2013, Chen et al., 2012, Danahay et al., 2015).

The parameters above will be further optimised in this chapter to create a viable lung organoid protocol.

3.1 Donor Characterisation NHBE and CHBE donors were age matched (62.75 vs 63.5 years, respectively) and purchased from Lonza. NHBE cell donors were never smokers while CHBE cell donors were all ex-smokers with an average 54.9 pack/years. None of the NHBE donors died from a lung-related illness. CHBE cell donors died at GOLD stage IV from COPD-related lung degeneration/emphysema. Half of NHBE donors were male compared with 75% of CHBE donors. All donors were Caucasian apart from one male donor (Tables 3.1.1 and 3.1.2).

Table 3.1.1-Nornal Human bronchial epithelial (NHBE) cell donor demographics: – Donors were age matched with CHBE cell donors being selected for age >40 years. NHBE donors were all never smokers. Tissue NHBE Smoking Pack Lot Acquisition Age Sex Race Packs/Day Donor Duration Years Number

1 0000382850 26993 60 F C 0 0 0

2 0000420970 27740 69 F C 0 0 0

3 0000495837 21954 69 M O 0 0 0

4 0000619261 31952 53 M C 0 0 0

Average ------62.75 ------0 0 0

Table 3.1.2- COPD Human bronchial epithelial (CHBE) cell donor demographics: Donors were age matched with NHBE with CHBE cell donors being >40 years. CHBE donors were all ex-smokers at GOLD stage IV. Tissue Smoking CHBE Packs/ Pack GOLD Lot Acquisition Age Sex Race Duration Donor Day Years Stage Number (years)

1 0000407341 27425 76 F C 30 1 30 IV

2 0000409276 27467 53 M C 27 2 54 IV

3 0000430905 27937 66 M C 48 1 48 IV

4 0000436083 28043 59 M C 35 2.5 87.5 IV

Average 63.5 35 1.625 54.9 IV

NHBE and CHBE cells are obtained from cadaveric donor lungs via bronchoscopy brushings of the epithelium and therefore may contain a mixed population of cells. To characterise donor cells to ensure that only basal epithelial cells were present, gene expression of the basal cell markers p63 and itga6 were quantified by QPCR (Figure. 3.1.1) and protein expression was shown by immunofluorescence for P63 and ITGA6 (Figures 3.1.2 and 3.1.3). NHBE cells expressed significantly higher levels of p63 and itga6. 100% of donor cells expressed P63 and ITGA6.

p63 itga6

1.8 * 1.8 ***

1.6 1.6

1.4 1.4

1.2 1.2

Ratio of p63/18s of Ratio 1.0 Ratio of itga6/18s of Ratio 1.0

0.8 0.8 NHBE CHBE NHBE CHBE Disease Status Disease Status

Figure 3.1.1- Quantitative PCR of basal cell markers in donor cells: Donor cells were seeded at 2500 cells/well and grown for a period of 4 days. RNA was isolated, reverse transcribed and cDNA was used for QPCR analysis. Unpaired t-test was performed. *p<0.05 and ***p<0.001.

Figure 3.1.2. Normal Human bronchial epithelial (NHBE) donor cell protein expression of basal cell markers: NHBE cells were seeded at 2500 cells/well and cultured for 4 days until confluent. They were then fixed with 4% paraformaldehyde in well and assayed with express P63 and ITGA6 antibodies. Cells expressed basal epithelial cell markers P63 (magenta) and ITGA6 (green). Images are representative of n=4 different donors.

Figure 3.1.3. COPD human bronchial epithelial (CHBE) donor cell protein expression of basal cell markers: CHBE cells were seeded at 2500 cells/well and cultured for 4 days until confluent. They were then fixed with 4% paraformaldehyde in well and assayed with express P63 and ITGA6 antibodies. Cells expressed basal epithelial cell markers P63 (magenta) and ITGA6 (green). Images are representative of n=4 different donors.

3.2 Developing NHBE and CHBE Bronchosphere Model Bronchosphere culture was based on a method reported by Danahay et. al. where bronchospheres were cultured in 100 wells of a 384 well plate and wells successfully producing spheres with lumens that had beating cilia were visualised using a light microscope counted in 384 well plates (n=3 repeats with donor 420970). Replication of the method described by Danahay et al. allowed for the generation of bronchospheres. However, 90% of wells did not produce bronchospheres. In wells where bronchosphere generation was perturbed, cells formed spheroids by day 5 but sank and attached to the bottom of the well by day 8. The attachment of spheroids to the plastic surface resulted in loss of spheroid structure and the formation of an epithelial cell monolayer by day 14 (Figure 3.2.1, n=3 using donor 420970).

Figure 3.2.1-Effect of plastic on spheroid development: Contact of spheroids with the plastic causes deformation into a monolayer. Images are representative of 3 images from 100 wells and from donor 420970. Several factors can account for spheroid migration and attachment to the bottom of the well. Loss of humidity due to evaporation through opening and closing of the incubator door has been linked to ALI culture failure and may cause bronchosphere degradation. Therefore, Microclime lids that keep plate humidity constant were tested. Whilst the use of Microclime plate lids brought success, the rate of bronchosphere generation across the plate increased to 60%, reproducible results were not obtained. Therefore, Corning ultra-low attachment (ULA) plates that contain a hydrophobic surface were used. This resulted in 100% bronchosphere generation across the plate. Microclime plate lids were still used to keep evaporation across the plate constant (Figure 3.2.2).

Bronchosphere culture was further optimised by determining the number of cells required for bronchosphere formation in 384 well plates. NHBE cells were seeded at 97

100, 350, 600 and 950 and fed at day 8 and 14 of culture for 20 days using donor 2, n=3 in preliminary experiments. Bronchospheres did not form below or above 600 cells/well. Cell densities over this figure aggregated too rapidly and formed large clumps that died after 8 days in culture not allowing for bronchosphere quantification. Cell densities <600 cells resulted in a lack of cellular migration and aggregation and remained as single cells. Single isolated cells failed to develop into bronchospheres therefore could not be quantified for ability to form bronchospheres (Figure 3.2.2, 100 and 350).

Figure 3.2.2 Cell Density Assay: Cell numbers in each well were increased from 100 to 950 cells (top left-hand corner) in 384 well plates. Images at 5x magnification scale bar is 100μm. Images are representative of 4 NHBE donors from Table 3.1.1. Images from NHBE (Donor 2 Table 3.1.1).

To determine the length of time required for bronchosphere formation, NHBE-B and CHBE-B were cultured in parallel. NHBE-B developed faster than CHBE-B cells (15.3 ± 0.7 vs 19.4 ± 0.4 days, n=4, p=0.4171) on average by 4.1 ± 0.8 days (Figure 3.2.3 A). Therefore, bronchospheres were cultured for 20 days from this point onward to ensure adequate formation for cells from all subjects.

After 20 days of culture both NHBE and CHBE cells generated similar numbers of bronchospheres in each well with NHBE cells generating 81.42 ± 4.804 and CHBE cells generating 77.08 ± 1.287 bronchospheres/well (Figure 3.2.3 B). However, lumen sizes in NHBE-B were lower than in CHBE-B 295.2 ± 9.29 vs 387.0 ± 34.1 (Figure 3.2.3 C and D).

A B

22 ** 100

20 90

18 80 16 70

Bronchosphere 14

# of Bronchospheres # of Development (Days) Development 60 12 NHBE CHBE NHBE CHBE Disease Status Disease Status

C D i

* 600

400

200 i

Lumen Diameter (µm) Diameter Lumen i 0

NHBE CHBE Disease Status

Figure 3.2.3 Bronchosphere development, Bronchospheres/well and Lumen sizes of bronchospheres from normal human bronchial epithelial cells (NHBE-B) and from bronchial HBEs from COPD patients (CHBE-B): Number of days elapsed for each bronchosphere to form/donor was counted A, mean of the number of bronchospheres in each of 3 wells from 4 patients B, mean of the diameter of the lumen of 3 largest bronchosphere from the same wells C. D Representative images of NHBE-B (i) and CHBE-B (ii) Unpaired t-test *p≤0.05, ***p≤0.001. NHBE and CHBE cells (600 cells/well) were seeded into 384 well plates and cultured over 20 days being fed on days 8 and 14. Cells or bronchospheres were fixed with 4% paraformaldehyde on days, 0, 2, 8, 14 and 20 (n=4 NHBE and CHBE donors).

Different bronchosphere luminal developmental stages were assessed by staining of the whole bronchospheres in situ with DAPI (blue) tumour protein 63 (P63, magenta), e-cadherin (E-CAD, green).

Both NHBE-B and CHBE-B initially formed by the autonomous aggregation of single P63 protein expressing cells into spheroids and subsequently merging of spheroids together to increase their size. Aggregation was achieved through the migration of single cells or spheroids towards each other. E-CAD protein expression was observed at day 2 when 2 or more cells aggregated together. NHBE spheroids began forming lumens at day 8 and finished lumen formation by day 14 shown by a lack of DAPI, P63 and E-CAD staining in the centre of the spheroids (Figure 3.2.4 A).

Luminogenesis was slower in CHBE spheroids, where DAPI staining was still observed in the centre of the spheroids at day 8, indicating the presence of cells. By day 14 DAPI+ cells were not observed in the central lumen of CHBE spheres (Figure 3.2.4 A and B). Interestingly, IF results showed that the expression of P63 protein expression was confined to the cells on the outer edge of the spheres even when DAPI+ CHBE cells were present in the centre of the spheroids as seen at day 8 (Figure 3.2.4 B). All donor spheres had fully formed lumens by day 20 and cells in the outer regions of the spheres remained positive for P63 protein.

100

A) Normal human bronchial epithelial bronchospheres (NHBE-B)

Development of normal healthy human bronchial epithelial bronchospheres (NHBE-B). See legend on next page.

101

B) COPD human bronchial epithelial bronchospheres (CHBE-B)

Figure 3.2.4 Development of normal healthy human bronchial epithelial (NHBE-B) and COPD human bronchial epithelial bronchospheres (CHBE-B) during 20 days of culture: A and B- NHBE or CHBE cells (n=4 donors) were seeded into 25% matrigel and fed with differentiation media on days 2, 8, and 14 for 20 days. 3 wells were fixed with 4% paraformaldehyde in well and pooled together on days 0, 2, 8, 14 and 20 of culture. Bronchospheres were assayed for E-cadherin (E-CAD, green) and tumour protein 63 (P63, magenta) protein. 4′,6-diamidino-2-phenylindole was used to stain DNA in all cells. P63 protein was detected in all donors from day 0 and E-CAD expression was detected at day 2 when 2 or more NHBE or CHBE cells aggregated. NHBE-B formed lumens by day 14 and CHBE-B formed lumens by day 20.

102

To confirm similarity with human airways, antibodies for goblet (MUC5AC), cilia (α-Tubulin or α-TUB) and basal cells (P63), were used to stain human bronchus 4 μM paraffin sections (n=3) to show airway pathology. The human airway epithelium demonstrated localisation of P63+ cells below secretory MUC5AC+ cells. α-TUB+ cilial staining was observed at the same level of MUC5AC+ cells at the luminal surface (Figure 3.2.5).

These features were subsequently assessed by IF in bronchospheres at day 0, 2, 8, 14 and 20 to show the timepoints at which ciliated and goblet cells developed in bronchospheres and whether bronchospheres were able to form the correct airway pathology at the end of 20 days of culture (Figure 3.2.6 A and B).

Figure 3.2.5 Immunofluorescence staining of human airways: 4μM healthy human airway paraffin sections were stained for goblet (mucin 5AC, MUC5AC), cilia (α-Tubulin, α-TUB) and basal epithelial cells (tumour protein 63, P63) to provide a comparison for bronchosphere differentiation. Staining is representative of n=3 sections. MUC5AC and α-TUB protein expression was not observed in NHBE-B and CHBE-B until spheroids had formed lumens (Figure 3.2.6 A and B). MUC5AC+ and α-TUB protein expression was observed in NHBE-B lumen at day 14 and continued to be detected until day 20 (n=4 donors) (Figure 3.2.6 A) as seen in airway bronchus staining (Figure 3.2.5). In contrast, MUC5AC and α-TUB protein expression was observed 6 days later in CHBE-B at day 20 of culture for all 4 donors (Figure 3.2.6 B). MUC5AC staining of CHBE-B at day 20 showed a region of densely stained luminal

103

MUC5AC that resembled a mucous plug like structure compared to NHBE-B small spots of MUC5AC were observed (Figure 3.2.6 B).

A) Normal Healthy human bronchial epithelial bronchospheres (NHBE-B)

Differentiated epithelial linneage of NHBE-B. See legend at bottom of (B) on next page for detail.

104

B) COPD human bronchial epithelial bronchospheres (CHBE-B)

Figure 3.2.6 Development of normal healthy human bronchial epithelial (NHBE-B) and COPD human bronchial epithelial bronchospheres (CHBE-B) during 20 days of culture: A and B- NHBE or CHBE cells (n=4 donors 3 pooled wells) were seeded into 25% matrigel and fed with differentiation media on days 2, 8, and 14 for 20 days. 3 wells were fixed with 4% paraformaldehyde in well and pooled together on days 0, 2, 8, 14 and 20 of culture. Bronchospheres were assayed for goblet cell by mucin 5 AC (MUC5AC, green) and ciliated cells by α-Tubulin (α-TUBLIN, red) protein expression. 4′,6 diamidino-2-phenylindole was used to stain DNA in all cells. α-TUB and MUC5AC protein was detected in all NHBE-B donors from day 14. Both markers were detected in all bronchospheres on day 20. NHBE-B and CHBE-B (n=4 donors respectively) were lysed in well, RNA was extracted and pooled and reverse transcribed into cDNA at day 0, 2, 8, 14 and 20 of

105

culture. foxj1, muc5ac and p63 gene expression levels were quantified by RT-QPCR to assess the timepoint of gene expression during NHBE-B and CHBE-B development.

Overall gene expression profile of NHBE-B and CHBE-B development mimicked IF data (Figure 3.2.7). p63 levels remained constant and at similar levels for both NHBE and CHBE over 20 days of culture (Figure 3.2.7).

NHBE-B and CHBE-B muc5ac gene expression was evident on day 8 of culture although MUC5AC protein expression in NHBE-B and CHBE-B was not detected until day 14 and 20 respectively. NHBE-B muc5ac gene expression remained constant throughout day 8-20 of culture. NHBE muc5ac was lower than CHBE-B expression at day 8 (278.5 ± 16.4 vs 497.5 ± 2.5). CHBE-B continued to be higher than NHBE-B expression at day 20 (213.9 ± 6.6 vs 149.1± 3.2) (Figure 3.2.6 B).

NHBE-B foxj1 gene expression (Figure 3.2.7 C) was detected on day 8 of culture whereas CHBE-B expression began at day 14. NHBE-B foxj1 gene expression was higher than CHBE-B at day 20 (195.0 ± 21.3 vs 136.1 ± 0.8).

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A B 300 250

200 200 150

100 100

tp63 tp63 Expression 50

muc5ac Expression muc5ac

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Figure 3.2.7- Gene expression of normal healthy human bronchial epithelial (NHBE-B) and COPD human bronchial epithelial bronchospheres (CHBE-B) during 20 days of culture: - NHBE or CHBE cells (n=4 donors) were seeded into 25% matrigel and fed with differentiation media on days 2, 8, and 14 for 20 days. Wells were lysed on days 0, 2, 8, 14 and 20 of culture, RNA was extracted, pooled, reversed transcribed and gene expression of ciliated cells by forkhead box j1 (foxj1) basal cells by tumour protein 63 (p63) and goblet cells by mucin 5 AC (muc5ac) was quantified by PCR. Results were normalised to 18s housekeeping gene and x by 100 to show relative expression. NHBE-B cells are shown in blue and CHBE-B cells are shown in red. Shown in mean ± SEM of at least 3 independent donors. p63 gene expression was detected from 0-20 of culture for all conditions whilst muc5ac for both conditions was detected from day 8 until day 20 of culture. gene expression of foxj1 started at day 8 and was detected until day 20. In comparison, foxj1 expression of CHBE-B was detected from on day 14-20.

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3.3 Developing a Chronic Smoking Bronchosphere Model One of the main causes of COPD is chronic cigarette smoke exposure. To assess the effect of cigarette smoke exposure, bronchosphere cultures were treated with differentiation media containing cigarette smoke condensate (CSC). To determine the optimal concentration of CSC required to treat bronchospheres, dose-response experiments were conducted using NHBE monolayer culture of 4 donors. cyp1a1 and cyp1b1 genes are up-regulated by aryl hydrocarbons within the CSC and their expression was used to assess the effect of CSC.

NHBE cells were treated with 0.01, 0.1, 1, 10, 100 and 1000 ng/ml of CSC in BEGM. cyp1a1 and cyp1b1 expression increased with increasing concentrations of CSC with the highest expression of cyp1a1 and cyp1b1 occurring at 1000 ng/ml (Figure 3.3.1 A and B).

MTT cell viability assay was conducted to determine NHBE viability after long term CSC treatment over 8 days. 0.01-1 ng/ml CSC treated NHBE cultures had >90% survival over 8 days. Cell survival dropped to ≤50% between 10-1000 ng/ml. The CSC concentration chosen to treat NHBE cells during bronchosphere development was therefore 1 ng/ml as it induced the highest cyp1a1 and cyp1b1 gene expression with a cell survival rate >90% (Figure 3.3.1).

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A CYP1A1 B CYP1B1 *** 30 20 *** *** *** *** 15 ***

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Cell Viability (% Control) Viability Cell 0 1 10 0.1 100 0.01 1000 DMSO Control CSC ng/ml

Figure 3.3.1 Cigarette smoke condensate (CSC) treatment of normal healthy human bronchial epithelial cells (NHBE) over 8 days of culture: NHBE cells were seeded at 2500 cells/well in bronchial epithelial cell medium supplemented with either 1 ng/ml DMSO or 0.01-1000ng/ml CSC. After 8 days wells were lysed, RNA extracted, pooled and reversed transcribed. Gene expression was quantified by QPCR for cytochrome P450 family 1 subfamily a member 1 (cyp1a1) (A) or cytochrome P450 family 1 subfamily b member 1 (cyp1b1) (B). cyp1a1 and cyp1b1 gene expression increases with increasing doses of CSC over 8 days but not in the absence of CSC. Therefore, gene expression was normalised to 0.01 ng/ml CSC. (C) Cell survival dropped by 50% at 10 ng/ml and further drops to 32% at 1000 ng/ml. Data are expressed as mean ± SEM of at least 3 independent donors. One-way ANOVA with Bonferroni correction was used to assess data. *p≤0.05, ***p≤0.001.

NHBE cells were seeded at 600 cells/well into 384 well plates and were fed with differentiation media supplemented with either 1 ng/ml of DMSO or CSC on day 8 and 14 of culture for 20 days and were hence termed DMSO-NHBE or CSC-NHBE. Cells

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or bronchospheres were fixed with 4% paraformaldehyde on days, 0, 8, and 20 (n=4 donors for both DMSO-NHBE-B or CSC-NHBE-B). As previously described in NHBE-B and CHBE-B, DMSO-NHBE and CSC-NHBE formed spheroids by autonomous aggregation of single P63 protein expressing cells forming spheroids. Subsequently spheroids merged together to increase their size. Aggregation was achieved through the migration of single cells or spheroids towards each other. DMSO-NHBE-B and CSC-NHBE-B cells both expressed E-CAD protein from day 2 onward, when 2 or more cells grouped together (Figure 3.3.2 A and B). E-CAD protein continued to be expressed throughout 20 days of culture between cells of either DMSO-NHBE-B or CSC-NHBE-B. P63 protein expression observed from day 0 in single cells continued to be expressed by the outer population of the spheroids as observed with NHBE and CHBE cells (Figure 3.2.4 A and B) and in spheres throughout the culture period until day 20 (Figure 3.3.2). DMSO-NHBE luminal development followed that of the NHBE-B timeline forming from day 8. CSC-NHBE lumen formation did not start at day 8 of culture with DAPI and E-CAD protein stained cell population was still visible in the sphere centre (Figure 3.3.2 B). DMSO-NHBE-B and CSC-HBE-B both had fully formed lumens by day 20 of culture as no DAPI and E-CAD stained cells were visible in the centre of the spheres (Figure 3.3.2 A and B). However, CSC-NHBE-B were not spherical in shape and displayed multiple lumens at day 20 of culture and therefore had an abnormal phenotype compared to NHBE-B and DMSO-NHBE-B controls that formed bronchospheres with a single fully inner lumen (Figure 3.2.6 A and 3.3.2 A).

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A) DMSO-treated normal healthy human bronchial epithelial bronchospheres (DMSO-NHBE-B)

Development of DMSO-NHBE-B. See legend at bottom of next page for full details.

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B) Cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial bronchospheres (CSC-NHBE-B)

Figure 3.3.2 Development of DMSO- or cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (DMSO-NHBE-B and CSC-NHBE-B respectively) during 20 days of culture: NHBE cells (n=4 donors 3 pooled wells) were seeded into 25% matrigel and fed with differentiation media supplemented with either DMSO (A) or 1 ng/ml CSC (B) on days 2, 8, and 14 for 20 days. 3 wells were fixed with 4% paraformaldehyde in well and pooled together on days 0, 8 and 20 of culture. Bronchospheres were assayed for E-cadherin (E-CAD, green) and tumour protein 63 (P63, magenta) protein. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain DNA in all cells. P63 protein was detected in all donors from day 0 and E-CAD expression was detected at day 2 when 2 or more NHBE or CHBE cells aggregated. DMSO-NHBE-B formed a single lumen by day 14 and CSC-NHBE-B formed multiple lumens by day 20.

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DMSO-NHBE-B and CSC-NHBE-B samples obtained at day 0, 8 and 20 of culture were assessed for goblet (MUC5AC) and ciliated (α-TUB) cells. As observed with NHBE-B and CHBE-B, MUC5AC and α-TUB protein expression was not observed in DMSO-NHBE-B until spheroids had formed lumens. DMSO-NHBE spheroids, followed the NHBE-B timeline for MUC5AC and α-TUB protein expression (Figure 3.2.6 A and 3.3.3 A). MUC5AC and α-TUB protein expression was observed in DMSO-NHBE-B lumen at day 14 and continued to be detected until day 20 (n=4 donors) (Figure 3.3.3 A) as seen in the human airway bronchus (Figure 3.2.5). In contrast, MUC5AC and α-TUB protein expression was not observed in CSC-NHBE-B lumen in any donor (Figure 3.3.3 B). Therefore, CSC-NHBE-B did not develop goblet or ciliated cells.

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A) DMSO-treated normal healthy human bronchial epithelial bronchospheres (DMSO-NHBE-B)

Full details are provided in the legend at the bottom of the next page.

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B) Cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial bronchospheres (CSC-NHBE-B)

Figure 3.3.3 Development of DMSO- or cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (DMSO-NHBE-B or CSC-NHBE-B respectively) during 20 days of culture: NHBE cells (n=4 donors representative of 3 pooled wells) were seeded into 25% matrigel and fed with differentiation media supplemented with either DMSO (A) or 1ng/ml CSC (B) on days 2, 8, and 14 for 20 days. 3 wells were fixed with 4% paraformaldehyde in well and pooled together on days 0, 8 and 20 of culture. Bronchospheres were assayed for goblet cell by mucin 5 AC (MUC5AC, green) and ciliated cells by α-Tubulin (α-TUB, red) protein expression. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain DNA in all cells. α-TUB and MUC5AC protein was detected in all DMSO-NHBE-B donors from day 14. In comparison expression of both markers were not detected for CSC-NHBE.

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DMSO-NHBE-B and CSC-NHBE-B (n=4 donors respectively) were lysed within the well, RNA extracted, pooled and reverse transcribed into cDNA at day 0, 8 and 20 of culture. foxj1, muc5ac and p63 gene expression levels were quantified by QPCR to assess the timepoints of gene expression during DMSO-NHBE-B and CSC-NHBE-B development. p63 gene expression was the highest at day 8 for both DMSO-NHBE-B and CSC-NHBE-B with DMSO-NHBE-B expression being lower than CSC-NHBE-B expression (189.7 ± 8.8 and 201.4 ± 11.9 respectively) (Figure 3.3.4 A). DMSO-NHBE-B and CSC-NHBE-B muc5ac gene expression was detected on day 8 of culture as previously seen in NHBE-B and CHBE-B profiling (Figure 3.2.7) even though no MUC5AC protein was detected in CSC-NHBE-B (Figure 3.3.3). DMSO-NHBE and CSC-HBE muc5ac gene expression levels were similar (DMSO-NHBE-B vs CSC-NHBE-B: 201.8 ± 12.1 and 200.9 ± 4.3 respectively) (Figure 3.3.4 B). The expression of foxj1 was evident on day 8 in DMSO-NHBE-B and was maintained until day 20 (Figure 3.3.4 C). In contrast, foxj1 expression was not detected until day 20 in CSC-NHBE-B (Figure 3.3.4 C) although no α-TUB protein expression was detected by IF at day 20 of culture (Figure 3.3.3). The expression of the foxj1 gene was higher in the DMSO-NHBE-B than CSC-NHBE-B cultures (212.3 ± 9.9 vs 144.2 ± 49.5) (Figure 3.3.4 C).

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Figure 3.3.4 Development of DMSO- or cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (DMSO-NHBE-B and CSC-NHBE-B respectively) during 20 days of culture: NHBE or CHBE cells (n=4 donors) were seeded into 25% matrigel and fed with differentiation media on days 2, 8, and 14 for 20 days. Wells were lysed on days 0, 8, and 20 of culture, RNA extracted, pooled and reverse transcribed. Gene expression representative of ciliated cells (forkhead box j1, foxj1) (A), basal cells (tumour protein 63, p63) (B) and goblet cells (mucin 5 AC, muc5ac) (C) was quantified using QPCR. Results were normalised to 18s housekeeping gene and multiplied by 100 to show relative expression. DMSO-HNBE-B are shown in orange and CSC-NHBE-B are in green. Results are presented as mean ± SEM of at least 3 independent donors. p63 gene expression was detected from 0-20 of culture whilst muc5ac for both treatments was detected from day 8 until day 20 of culture. gene expression of foxj1 was detected at day 8 and continued until day 20. In comparison, foxj1 expression in CSC-NHBE-B was only detected on day 20.

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3.4 Discussion The data in the present study showed that P63+ and ITGA6+ basal epithelial cells migrated towards each other to form spheroids. At the same time, they undergo mitotic division since the expression of epithelial markers such as E-CAD and α-TUB were observed. The cells at the centre of the spheroids lost P63 expression before the formation of a liquid filled lumen occurred. The inner lining differentiated into α-TUB+ ciliated and MUC5AC+ goblet cells with cells surrounding the differentiated inner lumen remaining P63+. These spheres containing a differentiated luminal were termed bronchospheres. Other studies using soft gel concentrations (<50% Matrigel) have reported similar cellular aggregation and spheroid formation as observed in this study compared to those using stiffer gel concentrations of >50% where cells do not migrate and form clonal cultures (Barkauskas et al., 2013, Tata et al., 2013, Chen et al., 2012, Danahay et al., 2015). As previously discussed, (Introduction Section 1.4), the foetal environment of the lung is not static, but dynamic, with epithelial cells migrating alone and collectively to allow for tubular extension and patterning of the lumen. The ability of spheroidal migration may be evidence of the collective migratory and pluripotent nature of basal epithelial cells.

The migratory nature of basal epithelial cells is also observed in ALI cultures post airlift when the culture is fully confluent (Park et al., 2015). However, the current studies report basal cell migratory behaviour under the context of cell jamming where cells are motile in a confluent population until a stabilising conformation in the population causes basal and lateral adhesional forces leading to cytoskeletal changes (Park et al., 2015). The difference in basal cell migratory properties seen during spheroid formation in this study is the continuous collective migration of a group of aggregate cells which is not modelled by ALI culture (Park et al., 2015).

Interestingly, during optimisation studies, where the initial seeding density was high (>600 cell/well), basal cell migration was increased, forming a small spheroid population often with 1-4 spheroids compared to 80+ bronchospheres observed in optimised wells. This suggests that there is an optimal cell population within a spheroid that allows for differentiation to occur and surpassing this population threshold leads to ‘spheroid catastrophe’ and cell death. From a tumour perspective these observations hold true where rapidly increasing tumour sizes often lead to increased 118

acidosis, necrosis and cellular starvation in the central cell populations compared to the outer cell population of the tumour (de Bruin et al., 2014). The rapid migration and spheroid formation may cause these factors to occur in the central populations of spheroids blocking progenitor differentiation (Figure 3.2.2).

Conversely where cell seeding was <600 cells/well basal cells could not aggregate and bronchosphere development was not observed. The data suggests that the lack of differentiation in this instance is due to the isolation of initiator basal cells and the early spheroids. Therefore, the study of cellular population thresholds in bronchosphere development may merit further study to understand the importance of population density in progenitor cell behaviour and differentiation. A limitation of this aspect of the study was that no in-depth analysis of the different cell types was performed as spheroids were cultured at different starting cell densities. Future studies should use single cell RNA-sequencing of bronchospheres and bioinformatic analysis of epithelial cell transcriptomic signatures over time to determine temporal changes in cell types. This will add greater confidence to the focused IF and RT-QPCR analysis performed here.

Optimisation studies showed that it is crucial that no contact between spheroids/bronchospheres and plastic surfaces occurs. Indeed, when spheroids/bronchospheres encountered plastic, the spheroidal structure was lost, and a monolayer was formed together with the dedifferentiation of specialised cells such as cilial cells. Airway epithelial cells have previously been shown to be plastic, dedifferentiating upon airway injury and redifferentiating during tissue healing (Crosby and Waters, 2010). However, once spheroids/bronchospheres had dedifferentiated and formed monolayers, cells did not aggregate into spheroids and differentiate again, rather remaining in their monolayer state. Cells seem to tend towards stiffer surfaces as evidenced by several protocols where single cell layers are grown out from tissue explants for example during airway smooth muscle isolation (Pang and Knox, 1997).

Gene expression of muc5ac was elevated several days before cellular differentiation was observed (Figures 3.2.4-3.2.7 and 3.2.3-3.3.3) which may indicate that mRNA levels may need to reach a certain threshold before development of structures such as mucous secreting vesicles or cilia can occur.

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No differentiation was detected using IF analysis of CSC-NHBE-B although muc5ac and foxj1 gene expression was detected at day 20. It is possible that factors within CSC may be inhibiting bronchosphere development post transcriptionally. It is also possible that the dose of CSC may be too high and that this may be inhibitory to bronchosphere development. The main driver(s) of CSC that cause airway remodelling in vivo are unknown and therefore any concentration applied in vitro is not comparable to the in vivo situation. Further extension of timepoints above 20 days and a full range of CSC concentrations may be necessary for the development of CSC-NHBE-B, as gene expression of foxj1 and muc5ac were observed at day 20 (Figures 3.2.3-3.3.3) (Schamberger et al., 2014). As detailed above in this discussion section, bioinformatic analysis of bronchospheres may determine the effects of CSC on epithelial cell differentiation in this model and how this relates to samples obtained from bronchial brushings of smokers and patients with COPD (Steiling et al., 2013, Campbell et al., 2012, Schembri et al., 2009, Carolan et al., 2009, Walters et al., 2014).

Previous studies in human lung basal epithelial cells have observed the increase of proteins such as EGF due to cigarette smoke, and a shift in basal cells to a more squamous EMT-like phenotype (Shaykhiev et al., 2013) which may also explain the lack of differentiation (Yang et al., 2017). The presence of an EMT-like phenotype could also explain the presence of multiple lumens. The presence of multiple lumens in CSC-NHBE-B may indicate a dysfunction in polarity proteins, which have previously been observed in MCDK cysts with mutant polarity complexes as previously discussed (Introduction section 1.2.4.2). Further research will be required to elucidate the mechanisms causing the lack of differentiation in CSC-NHBE-B.

In summary the current chapter has demonstrated that the airway epithelium can be modelled in 3D and is able to replicate features of the airway and airway disease. The models show developmental events in lung epithelium formation, which is poorly understood and effectively shows the effect of a noxious agent on progenitor cell programming. Several pathways may be deregulated in bronchospheres such as polarity complexes as evidenced by the presence of multiple lumens in CSC-NHBE-B (sections 3.2.3-3.3.3) and the Notch, Wnt and SOX pathways. These pathways require further elucidation to truly understand the model.

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4 Chapter 4

Global Expression of Transcripts in Bronchospheres

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4.0 Introduction The development of the lung epithelium is dependent upon signalling by multiple gene pathways. However, the perturbations of these pathways may result in the dysregulation of the control of epithelial progenitor cell differentiation resulting in changes in airway epithelial cell populations (Shaykhiev and Crystal, 2014b). This may lead to the formation of airway morphologies including those seen in airway diseases such as COPD. Recent evidence has suggested that chronic injury to the airway epithelium in COPD may cause basal epithelial cells to be reprogrammed resulting in atypical regeneration of the airway (Shaykhiev and Crystal, 2014a). The airway remodelling observed in COPD patients includes goblet cell hyperplasia and ciliated hypoplasia (Olea et al., 2011, Shaykhiev and Crystal, 2014a). The NHBE and CHBE basal epithelial cells in Chapter 3 were shown to form bronchospheres that replicate lung epithelial morphology observed in never smokers and COPD human donors. This supports the hypothesis that progenitor cell differentiation pathways may be reprogrammed in disease.

Whilst the dysregulation of differentiation pathways including Notch and Wnt are involved in COPD pathogenesis (Shi et al., 2017, Zong et al., 2016), the genes involved in COPD pathogenesis and in basal epithelial cell differentiation are poorly studied. Genetic studies have not been able to identify the drivers of airway remodelling mainly due to a lack of proper models relating to the complex architecture of the airway epithelium (Prakash et al., 2017, Berndt et al., 2012) and due the lack of knowledge of lung development and repair. These factors have been a barrier to the identification of novel targets that would stop or slow COPD progression.

Furthermore, currently there are no CSC-treated- or CHBE-derived bronchosphere models therefore genes involved in CHBE bronchosphere formation have not been explored. Therefore, RNA-seq analysis was used to explore changes in gene expression over the course of bronchosphere culture to elucidate the transcriptional landscape that generated the NHBE-B and CHBE-B phenotypes. In addition, the effect of CSC on NHBE-B transcriptional profiles over 20 days was analysed.

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4.1 Quality Control of Bronchosphere RNA RNA quality and quantity were measured using a bioanalyser and the RNA integrity number (RIN) threshold was set at 7 before PolyA-RT sequencing. Samples that did not meet RIN threshold were excluded and not analysed. All NHBE and CHBE samples were above the threshold. However, Day 8 CSC-NHBE and DMSO-NHBE samples had RIN values less than 7 and were excluded from PolyA-RT sequencing. The sequencing was performed by Novartis (Boston, MA, USA) using an in-house platform. No further details of methodology are provided. The analysis was performed by Dr Vera Ruda.

Once PolyA-RT sequencing was complete, the Fastqc program was used to further analyse the quality of the data. The reads were aligned by Dr Vahid Elyasigomari at the Data Sciences Institute at Imperial College London.

4.2 Differences between COPD (CHBE-B) and normal healthy bronchospheres (NHBE-B) at day 0 NHBE and CHBE cells were seeded in 5% matrigel on top of 25% matrigel in differentiation media and incubated at 37°C for 2 hours. Other NHBE cells were cultured in 5% matrigel media supplemented with 1ng/ml CSC before being incubated in the same manner for 2 hours (CSC-NHBE). This enabled the cells to rest after cell processing (trypsinisation) and any cell-cell-matrix communication to begin before being lysed for RNA extraction. This was termed baseline or day 0 of culture. To explore the effects of the bronchosphere culture environment on epithelial cells, CHBE and CSC-NHBE gene expression was compared to NHBE cellular expression at baseline (day 0, Table 4.2.1 and 4.2.3) using the STAR analysis package to align reads and differentially expressed genes (>2-fold difference, adj. p value <0.05). At baseline, 1735 genes were 2-fold differentially expressed between CHBE and NHBE cells with an adjusted p value of <0.05. Of these, 663 genes were up-regulated, and 1072 genes were down-regulated. A heat map of the top 30 up- and down- regulated genes demonstrated a clear distinction between CHBE and NHBE cells with the CSC-NHBE cells clustered between these extremes (Figure 4.2.1).

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Figure 4.2.1-Baseline differential gene expression: Heat map showing 30 top differentially expressed genes at Day 0 of bronchosphere culture. Overall, there were 1735 differentially expressed genes between COPD human bronchial epithelial cells (CHBE) and epithelial cells from normal healthy subjects (NHBE) cells. The top 15 significantly up-regulated genes in COPD compared to normal healthy bronchospheres (Table 4.2.1) included genes such as caspase 5 (casp5), Adenosine Deaminase, RNA-Specific, B2 (adarb2), S100 Calcium Binding Protein P (s100p), Galactose-3-O-Sulfotransferase 1 (galst1) and miR3189 which are involved in a number of COPD-relevant processes including cell proliferation and death, cell cycle progression and xenobiotic metabolism.

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In contrast, several genes were significantly down-regulated in COPD compared to normal healthy bronchospheres and the top 15 most down-regulated genes included X Inactive Specific Transcript (xist), Kinesin Family members (kif14, -18B and -20A), G2 and S-phase expressed 1 (gtse1) and the proliferation marker, marker of proliferation ki67 (ki67). These genes are involved in microtubule formation and function and epigenetic gene suppression (Table 4.2.1). 5 up-regulated and 1 down regulated transcript could not be identified within the top 15 differentially expressed genes between CHBE-B compared to normal healthy NHBE-B. This suggests that there may be other novel effectors that may be important in the regulation/differentiation of healthy versus COPD bronchospheres. These non- ascribed genes were excluded from pathway analysis (Table 4.2.1). The most significantly up and down-regulated genes, ranked by adjusted p-value (Table 4.2.2), included genes such as follistalin like 1 (fstl1), collagen and ECM binding domain 1 (ccbe1) and mmp9. fstl1 is a BMP4 antagonist and is required for correct lung development. Its deletion results in embryonic lethality in mice (Li et al., 2016). ccbe1 and mmp9 are involved in migration and extracellular matrix remodelling (Li et al., 2018, Buro-Auriemma et al., 2013, Atkinson and Senior, 2003). ccbe1 has been shown to be down regulated in basal cells of smokers (Buro-Auriemma et al., 2013) (Table 4.2.2).

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Table 4.2.1- Top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) and bronchospheres derived from normal healthy human epithelial (NHBE) cell (NHBE-B) ranked according to log2 fold-change at day 0 of culture: Top 15 highly expressed transcripts shown in red from 663 significantly differentially expressed genes and the bottom 15 least expressed transcripts shown in blue out of 1,072 significantly repressed genes. na=unidentifiable transcript sequences.

Up- and Down-regulated genes in COPD bronchospheres (CHBE-B) day 0 versus bronchospheres from healthy subjects (NHBE-B) at Day 0 Log Fold Log Fold Index Genes 2 Adjusted p-value Index Genes 2 Adjusted p-value Increase Decrease 1 casp5 7.731924 0.0001371 1 na -11.8908 3.61E-10 2 na 7.449222 0.0468832 2 mcoln2 -9.83791 3.73E-13 3 na 7.136458 1.48E-33 3 xist -9.5395 2.90E-05 4 na 7.055986 5.20E-121 4 kif18b -9.36826 2.97E-12 5 adarb2 6.913382 1.38E-06 5 spc25 -9.04584 5.35E-10 6 mir3189 6.742092 2.78E-05 6 dlgap5 -8.93824 6.33E-18 7 gpnmb 6.719713 1.15E-35 7 38047 -8.87238 1.19E-07 8 tmem59l 6.347585 1.44E-08 8 nek2 -8.74838 9.40E-30 9 na 6.106065 0.0001318 9 loc400655 -8.52287 2.19E-07 10 s100p 6.051572 6.57E-16 10 ki67 -8.46366 1.37E-36 11 na 6.048881 8.27E-06 11 gtse1 -8.41653 5.78E-14 12 gal3st1 5.927423 7.18E-05 12 sgo1 -8.10861 4.52E-19 13 na 5.91931 5.65E-05 13 kif20a -8.09895 1.43E-13 14 lingo2 5.907918 3.41E-10 14 kif14 -8.08707 9.85E-35 15 clgn 5.87937 0.0003926 15 hjurp -7.97516 1.30E-14

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Table 4.2.2- Top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) and bronchospheres derived from normal healthy human epithelial (NHBE) cell (NHBE-B) ranked according to adjusted p-value change at day 0 of culture: Up- and down regulated genes out of 1735 differentially expressed genes in CHBE-B day 0 versus NHBE-B day 0 ranked according to lowest adjusted p-value. na=unidentifiable transcript sequences.

Up- and Down-regulated genes in COPD bronchospheres (CHBE) day 0 versus bronchospheres from normal healthy subjects (NHBE) at Day 0 Log Fold Index Genes 2 Adjusted p-value Increase 1 na -4.67655 5.20E-121 2 fstl1 -3.56217 2.57E-63 3 ccbe1 -2.49604 3.84E-52 4 col8a1 2.474704 1.54E-40 5 mmp9 -2.90034 2.94E-40 6 depdc1 -3.58512 8.60E-40 7 tmprss4 -3.34425 2.24E-37 8 ki67 -5.28806 1.37E-36 9 top2a -2.68864 1.37E-36 10 cdkn3 -2.17122 1.54E-36 11 cep55 -2.1118 3.42E-36 12 gpnmb 2.972101 1.15E-35 13 kif14 -3.69942 9.85E-35 14 hmmr 7.449222 1.13E-34 15 ctnnal1 2.374202 3.05E-34

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Analysis of differentially activated pathways in CHBE-B at day 0

Pathways enriched in these all differentially expressed up- and down-regulated gene sets were examined using the online analysis tool Reactome 2016 (https://www.reactome.org) in EnrichR (http://amp.pharm.mssm.edu/Enrichr/), (Table 4.2.3). The top significantly up-regulated pathway in CHBE-B compared with NHBE-B at baseline was Diseases of Glycosylation with most up-regulated pathways involved in glycosylation, particularly of mucus, matrix biology and in protein glycolysis (Table 4.2.3).

The top significantly down-regulated pathway was cell cycle pathway with most down regulated pathways involving the down-regulation of mitosis and cell division (Table 4.2.3).

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Table 4.2.3-Pathway analysis of all the genes increased or decreased by ≥2-fold in COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) and bronchospheres derived from normal healthy human epithelial (NHBE) cell (NHBE-B) at day 0 of culture: The top 10 pathways were enriched for the 663 up-regulated genes (shown in red) whilst the bottom 10 down-regulated pathways using 1072 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR.

CHBE Day 0 vs NHBE Day 0 Pathway Analysis of Down-regulated CHBE Day 0 vs NHBE Day 0 Pathway Analysis of Up-regulated Genes Genes Adjusted Index Name P-value Index Name P-value Adjusted p-value p-value 1 Diseases of glycosylation 3.71E-07 0.000236 1 Cell Cycle 3.01E-58 2.70E-55 Defective B3GALT6 causes 2 9.4E-05 0.01301 2 Cell Cycle, Mitotic 9.64E-53 4.33E-50 EDSP2 and SEMDJL1 Defective B3GAT3 causes 3 9.4E-05 0.01301 3 M Phase 5.24E-26 1.18E-23 JDSSDHD Defective B4GALT7 causes EDS, 4 9.4E-05 0.01301 4 Mitotic Prometaphase 3.11E-29 9.32E-27 progeroid type Resolution of Sister 5 O-linked glycosylation 0.000123 0.01301 5 5.32E-25 9.55E-23 Chromatid Cohesion 6 O-linked glycosylation of mucins 0.000276 0.02191 6 Cell Cycle Checkpoints 4.38E-19 4.92E-17 Diseases associated with O- Mitotic Metaphase and 7 0.000178 0.01612 7 5.14E-19 5.13E-17 glycosylation of proteins Anaphase A tetrasaccharide linker sequence Signalling by Rho 8 0.000458 0.02656 8 2.21E-19 2.84E-17 is required for GAG synthesis GTPases 9 Dermatan sulfate biosynthesis 0.000122 0.01301 9 Mitotic Anaphase 2.86E-18 2.34E-16 Diseases associated with 10 0.000458 0.02656 10 RHO GTPase Effectors 1.81E-18 1.62E-16 glycosaminoglycan metabolism

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4.3 Effect of Bronchosphere Culture on NHBE and CHBE bronchospheres at Day 8 The transcriptomic profiles of NHBE-B and CHBE-B at day 8 were compared to their respective samples at day 0. 3297 genes were differentially expressed in NHBE-B and 3741 genes were differentially expressed in CHBE-B after 8 days. A heat map showing the top 30 differentially expressed genes is shown in Figure 4.3.1.

Figure 4.3.1-Differential gene expression after 8 Days of bronchosphere culture: Heat maps showing the 30 top differentially expressed of genes at Day 8 of bronchospheres derived from normal human bronchial epithelial (NHBE) cells (NHBE-B) and COPD bronchial epithelial (CHBE) cells (CHBE-B). Overall, 3297 and 3741 genes were differentially expressed between NHBE-B and CHBE-B at day 8 of culture compared with day 0 respectively.

Analysis of differentially expressed genes in bronchospheres from normal subjects at day 8

The top 15 significantly up- and down-regulated genes in NHBE-B at day 8 by fold-change are shown in Table 4.3.1. These include the cell-surface receptor for stem cell factor (kit), bone morphogenetic protein 3 (bmp3), glutathione S-transferase alpha 1 and 2 (gsta1 and -2) and polymeric immunoglobulin receptor (pigr). These 130

are involved in epithelial cell differentiation and proliferation and oxidant defence. In contrast, genes involved in immune responses, metabolic changes and inhibition of Wnt signalling were highly down-regulated after 8 days of culture including granulocyte macrophage-colony stimulating factor (csf2), interleukin 1 receptor 2 (il1r2), galactosidase beta 1 like 3 (glb1l3), the proto-oncogene fosb (fosb) and dickkopf Wnt signalling pathway inhibitor 2 (dkk2) (Table 4.3.1).

Most significantly up- and down-regulated genes ranked by adjusted p-value (Table 4.3.2) included genes such as solute carrier family 34 member 2 (slc34a2), pigr and the proto-oncogene FosB (fosb). slc34a2 and pigr were both up regulated and are involved in the transport of immune globin A and sodium across epithelial cell membranes respectively. fosb was down-regulated and has been shown to be important in regulation of cell proliferation, differentiation, apoptosis and transformation (Table 4.3.2).

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Table 4.3.1- Top differentially expressed genes between normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 8 and NHBE at day 0 ranked according to log2 fold-change: Top 15 highly expressed transcripts shown in red from 2,183 significantly differentially expressed genes and the bottom 15 least expressed transcripts shown in blue out of 1,114 significantly repressed genes. na=unidentifiable transcript sequences.

NHBE-B Day 8 vs NHBE Day 0 Log Fold Log Fold Index Genes 2 Adjusted p-value Index Genes 2 Adjusted p-value Increase Decrease 1 hla-dra 11.38154 1.49E-59 1 csf2 -8.5241 7.86E-19 2 na 10.16061 6.87E-13 2 dkk2 -8.04775 7.29E-12 3 kit 10.08586 2.89E-18 3 glb1l3 -7.37936 1.25E-48 4 plekhg7 10.08379 1.39E-18 4 fosb -7.35619 1.23E-233 5 gsta1 10.01395 1.15E-38 5 gpr141 -7.2938 3.42E-10 6 stath 9.932595 2.28E-12 6 38047 -7.20227 1.21E-07 7 znf648 9.859507 4.47E-19 7 na -7.15083 2.36E-10 8 gsta2 9.461031 1.25E-17 8 linc01704 -6.97791 2.70E-12 9 fmo6p 9.445164 2.95E-17 9 na -6.85685 1.41E-08 10 plekhs1 9.421579 2.93E-132 10 slc8a1 -6.8086 1.56E-61 11 pigr 9.387958 4.80E-292 11 na -6.67764 5.53E-08 12 bmp3 9.337522 2.65E-38 12 pappa2 -6.63419 1.36E-13 13 capn13 9.325849 3.43E-122 13 na -6.5864 9.32E-05 14 slc34a2 9.316437 10E-6 14 il1r2 -6.51826 2.54E-12 15 atoh8 9.244636 1.00E-75 15 ccbe1 -6.51552 6.88E-58

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Table 4.3.2-Up and down regulated genes in normal human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 8 versus NHBE at day 0 of culture: Up- and down regulated genes out of 2,297 differentially expressed genes in NHBE-B at day 8 versus NHBE-B at day 0 ranked according to lowest adjusted p-value. Where no p-value is given p<10-300. na=unidentifiable transcript sequences. na=unidentifiable transcript sequences. Up- and Down-regulated genes in normal healthy bronchospheres (NHBE-B) at day 8 versus day 0

Index Genes Log2 Fold Increase Adjusted p-value 1 slc34a2 9.316437 <10E-300 2 pigr 9.387958 4.80E-292 3 fosb -7.35619 1.23E-233 4 loxl4 8.159726 1.20E-221 5 tmprss4 5.388135 1.98E-205 6 ntn1 5.718611 4.08E-204 7 tjp3 7.132245 4.08E-204 8 bcas1 7.651701 1.22E-203 9 vtcn1 7.460722 9.95E-199 10 slc44a4 6.875745 2.07E-192 11 tcn1 8.386048 9.05E-176 12 serpinb4 5.74227 1.88E-169 13 ppm1h 7.345284 2.88E-169 14 aldh1a1 6.394266 1.74E-165 15 muc20 7.683426 6.26E-164

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Analysis of differentially activated pathways in NHBE-B at day 8

Pathway analysis of the top 15 up- and down-regulated genes at day 8 showed one pathway that was significantly associated with up-regulated genes and no significantly down-regulated pathways.

Pathway analysis of all differentially up-regulated genes in NHBE-B at day 8 highlighted pathways associated with metabolism particularly those linked with oxidative stress and biological oxidation. Other pathways included those associated with protein glycosylation such as O-linked glycosylation (Table 4.3.3).

Down-regulated pathways were those associated with cell division, proliferation and mitosis (Table 4.3.3).

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Table 4.3.3-Pathway analysis of all the genes increased or decreased by ≥2-fold in normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 8 compared to day 0 of culture: The top 10 pathways were enriched for the 2,183 up-regulated genes (shown in red) whilst the bottom 10 down-regulated pathways using 1,114 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR. na=unidentifiable transcript sequences.

NHBE-B Day 8 vs Day 0 Pathway Analysis of Up-regulated Genes NHBE-B Day 8 vs Day 0 Pathway Analysis of Down-regulated Genes Adjusted Adjusted Index Name P-value Index Name P-value p-value p-value Phase 1 - Functionalization of 1 8.37E-13 8.54E-10 1 Cell Cycle 5.71E-33 4.97E-30 compounds 2 Biological oxidations 2.88E-12 1.47E-09 2 Cell Cycle, Mitotic 9.40E-30 4.09E-27 3 Fatty acids 1.27E-09 2.59E-07 3 Mitotic Prometaphase 2.96E-24 8.59E-22 Resolution of Sister Chromatid 4 Xenobiotics 3.86E-10 9.85E-08 4 2.78E-22 6.06E-20 Cohesion Cytochrome P450 - arranged 5 3.07E-10 9.85E-08 5 M Phase 7.44E-17 1.08E-14 by substrate type 6 Arachidonic acid metabolism 1.26E-07 2.15E-05 6 RHO GTPases Activate Formins 4.08E-17 7.10E-15 Diseases associated with Mitotic Metaphase and 7 3.58E-07 5.22E-05 7 5.72E-14 6.22E-12 O-glycosylation of proteins Anaphase 8 O-linked glycosylation 8.43E-07 0.000108 8 RHO GTPase Effectors 3.23E-14 4.02E-12 Transmembrane transport of 9 6.36E-06 0.000564 9 Separation of Sister Chromatids 2.31E-13 2.15E-11 small molecules Defective GALNT12 causes 10 4.52E-06 0.000461 10 Mitotic Anaphase 2.74E-13 2.17E-11 colorectal cancer 1 (CRCS1)

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Analysis of differentially expressed genes in bronchospheres from COPD subjects at day 8

The 15 top up-regulated genes in CHBE bronchospheres at day 8 compared to day 0 are shown in Table 4.3.4. The genes include the flavin containing monooxygenases (fmo2) and (fmo6p), gsta1 and -2, secretoglobin family 1A member 1 (scgb1a1), lysyl oxidase like 4 (loxl4), metalloproteinase 13 (mmp13) and annexin 10 (anxa10). fmo2, fmo6p, loxl4, gsta1 and -2 are all found in oxidative pathways. mmp13 with loxl4 being crucial for the formation of connective tissue with mmp13 important for remodelling of ECM (Table 4.3.4).

Down-regulated genes included, chaC glutathione specific gamma-glutamylcyclotransferase 1 (chac1), fosb, forkhead box a2 (foxa2), interleukin 1 receptor like 1 (il1rl1) and interleukin 13 receptor subunit alpha 2 (il13ra2). chac1 glutothione regulation and participates in oxygen balance. chac1 and foxa2 are both involved in cellular differentiation and transformation. Il1rl1 and il13ra2 are involved in inflammatory pathways (Table 4.3.4).

2 up-regulated transcripts were not assigned (NA) whilst 5/15 down-regulated transcripts were unassigned indicating that other genes and pathways in the future may be assigned to a role in the differentiation of COPD bronchospheres at this time point. Non-assigned transcripts were not used to define pathways (Table 4.3.4).

Some genes were highly significantly differentially expressed at day 8 in CHBE-B as well as in NHBE-B when ranked according to adjusted p value. These included loxlp4, ntn1 and pigr which are associated with immune regulation, cell migration during development and connective tissue biogenesis and the cross-linking between collagen and elastin. Other up-regulated genes such as dusp1 and pbx1 were genes involved in cellular differentiation (Table 4.3.5)

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Table 4.3.4- Top differentially expressed genes between COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) at day 8 versus day 0 ranked according to log2 fold-change: Top 15 highly expressed transcripts shown in red from 2,788 significantly differentially expressed genes and the bottom 15 least expressed transcripts shown in blue out of 953 significantly repressed genes. na=unidentifiable transcript sequences.

CHBE-B Day 8 vs Day 0

Index Genes Log2 Fold Increase Adjusted p-value Index Genes Log2 Fold Decrease Adjusted p-value 1 fmo2 9.732216 7.79E-103 1 pappa2 -8.86017 3.45E-37 2 atoh8 9.429589 2.64E-87 2 fosb -8.25919 1.01E-188 3 gsta2 9.126635 4.87E-17 3` linc02261 -8.25406 2.67E-13 4 na 9.061233 3.06E-102 4 camp -7.5961 5.18E-20 5 fmo6p 9.048265 1.48E-16 5 trib3 -7.55944 6.81E-176 6 islr 8.81973 2.98E-16 6 na -7.48786 2.68E-10 7 scgb1a1 8.678072 1.48E-104 7 na -7.28675 4.43E-09 8 lrrc32 8.555656 9.83E-11 8 na -7.25784 5.31E-87 9 loxl4 8.499295 <10E-300 9 na -6.90887 3.74E-09 10 hoxc13-as 8.418218 2.50E-12 10 na -6.89073 6.13E-13 11 na 8.378218 1.60E-13 11 il1rl1 -6.77692 4.83E-34 12 mmp13 8.374066 4.38E-63 12 il13ra2 -6.76704 4.83E-40 13 anxa10 8.337114 9.52E-89 13 erich2 -6.46787 3.64E-36 14 mir3142hg 8.299689 2.50E-14 14 chac1 -6.38361 4.03E-207 15 gsta1 8.274987 7.21E-17 15 foxa2 -6.34078 4.49E-144

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Table 4.3.5- Up and down regulated genes in COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) at day 8 versus day 0: Up- and down regulated genes out of 3,741 differentially expressed genes in CHBE-B day 8 versus NHBE-B day 0 ranked according to lowest adjusted p-value. Adjusted p values are all p<10-300. na=unidentifiable transcript sequences. Up- and Down-regulated genes in COPD bronchospheres (CHBE-B) at day 8 versus day 0 Log Fold Index Genes 2 Adjusted p-value Increase 1 pbx1 3.121977 <10E-300 2 pigr 7.75294 <10E-300 3 odc1 -3.86545 <10E-300 4 dhrs9 6.619336 <10E-300 5 sidt1 3.972265 <10E-300 6 gabrp 8.037103 <10E-300 7 dusp1 -4.48803 <10E-300 8 adgrf1 3.870389 <10E-300 9 upp1 -4.98117 <10E-300 10 adam28 4.995346 <10E-300 11 na 2.70904 <10E-300 12 loxl4 8.499295 <10E-300 13 mettl7a 6.622734 <10E-300 14 stra6 7.990024 <10E-300 15 ntn1 4.973528 <10E-300

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Analysis of differentially activated pathways in CHBE-B at day 8

Pathway analysis indicated that the top 10 significantly up-regulated pathways (Table 4.3.6) involved activation of key pathways associated with the regulation of cellular oxidation, ECM organisation and regulation and the release of matrix degrading enzymes.

One pathway, amino acid transport across the plasma membrane was significantly associated with down-regulated genes in COPD bronchospheres at day 8. Non-significant pathways included immune signalling, responses to growth factor stimulation and oxidant responses to infection (Table 4.3.6).

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Table 4.3.6-Pathway analysis of all the genes increased or decreased by ≥2-fold in COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) at day 8 compared to day 0: The top 10 pathways were enriched for the 2,783 up-regulated genes (shown in red) whilst the bottom 10 down- regulated pathways using 953 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR.

CHBE-B Day 8 vs CHBE-B Day 0 Pathway Analysis of Up-regulated CHBE-B Day 8 vs CHBE-B Day 0 Pathway Analysis of Down- Genes regulated Genes Adjusted Adjusted Index Name P-value Index Name P-value p-value p-value Amino acid transport across the 1 Biological oxidations 3.60E-09 3.82E-06 1 6.61E-06 0.004681 plasma membrane Phase 1 - Functionalization of PERK regulates gene 2 2.71E-07 0.000144 2 3.17E-05 0.01122 compounds expression 3 Mitotic Prometaphase 4.70E-07 0.000166 3 ATF4 activates genes 0.000151 0.03574 Resolution of Sister Chromatid Amino acid and oligopeptide 4 1.22E-06 0.000323 4 0.000221 0.03907 Cohesion SLC transporters Amino acid synthesis and 5 Extracellular matrix organization 1.92E-06 0.000407 5 interconversion 0.001333 0.1888 (transamination) Unfolded Protein Response 6 Glucuronidation 2.39E-06 0.000423 6 0.002521 0.2506 (UPR) Defective B3GALT6 causes 7 Fatty acids 1.39E-05 0.002102 7 0.003379 0.2506 EDSP2 and SEMDJL1 Defective B3GAT3 causes 8 Arachidonic acid metabolism 3.85E-05 0.004542 8 0.003379 0.2506 JDSSDHD Degradation of the extracellular Defective B4GALT7 causes 9 4.59E-05 0.004869 9 0.003379 0.2506 matrix EDS, progeroid type Defective GALNT12 causes Chondroitin sulfate/dermatan 10 7.38E-05 0.006535 10 0.006639 0.2888 colorectal cancer 1 (CRCS1) sulfate metabolism

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Comparison of differentially expressed genes in bronchospheres derived from normal healthy and COPD subjects at day 8

A comparison of the top 15 up- and down-regulated genes in NHBE and CHBE bronchospheres at day 8 compared to day 0 shows some similarities and some differences (Table 4.3.7). There were greater similarities in the up-regulated genes than in the down-regulated genes. For example, fmo and gsta family and atoh8 genes were all up-regulated in both CHBE and NHBE bronchospheres. In contrast, the overlap between down-regulated genes was with fosB only although different interleukin receptor genes (il1r2 versus il1rl1 and il13ra2) were down regulated in both NHBE and CHBE bronchospheres (Table 4.3.7).

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Table 4.3.7- Top differentially expressed genes between COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) between day 8 and day 0 compared to the top differentially expressed genes in normal healthy human bronchial epithelial (NHBE) cell derived bronchospheres (NHBE-B) between day 8 and day 0 ranked according to log2 fold-change: Top 15 highly expressed transcripts shown in red and the bottom 15 least expressed transcripts shown in blue. na=unidentifiable transcript sequences.

Up-regulated genes between day 8 and day 0 Down-regulated genes between day 8 and day 0 Index NHBE CHBE Index NHBE CHBE 1 hla-dra fmo2 1 csf2 pappa2 2 na atoh8 2 dkk2 fosb 3 kit gsta2 3 glb1l3 linc02261 4 plekhg7 na 4 fosb camp 5 gsta1 fmo6p 5 gpr141 trib3 6 stath islr 6 38047 na 7 znf648 scgb1a1 7 na na 8 gsta2 lrrc32 8 linc01704 na 9 fmo6p loxl4 9 na na 10 plekhs1 hoxc13-as 10 slc8a1 na 11 pigr na 11 na il1rl1 12 bmp3 mmp13 12 pappa2 il13ra2 13 capn13 anxa10 13 na erich2 14 slc34a2 mir3142hg 14 il1r2 chac1 15 atoh8 gsta1 15 ccbe1 foxa2

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4.4 Effect of Bronchosphere Culture on NHBE and CHBE bronchospheres at day 20 compared to day 0

Figure 4.4.1-Differential gene expression after 20 Days of bronchosphere culture: Heat maps showing the 30 top differentially expressed of genes at Day 20 of bronchosphere culture for normal human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) and COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B). Overall, 4469 and 4438 were differentially expressed genes between NHBE-B and CHBE-B at day 8 of culture and NHBE at-B at day 0 respectively. Goblet cell markers muc5ac and BPI fold containing family B member 1 (bpifb1) as well as the club cell marker scgb3a1 and the ciliated cell master control gene foxj1 together with the cilial genes family with sequence similarity 92 member B (fam92b) and sperm associated antigen 6 (spag6) whose transcripts were detected at day 20 in NHBE-B culture and possibly reflect changes in cellular phenotypes (Table 4.4.1). SPAG6 has been previously shown to be important in cilial length and beat frequency and is crucial for cilial development.

Ranking genes by adjusted p-value (Table 4.4.2) identified genes that were up- regulated in NHBE-B at day 20 including fosb, ntn1, pigr and slc34a2. Genes that were expressed differently from NHBE-B at day 8 included mucin 4 (muc4). The up- regulated genes mucin 4 (muc4) and mucin 20 (muc20) both participate in the O-linked glycosylation pathway (Table 4.4.2).

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Table 4.4.1- Top differentially expressed genes between normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 20 compared to day 0 ranked according to log2 fold-change: Top 15 highly expressed transcripts shown in red from 3,301 significantly differentially expressed genes and the bottom 15 least expressed transcripts shown in blue out of 1,168 significantly repressed genes. na=unidentifiable transcript sequences. NHBE-B Day 20 vs Day 0

Index Genes Log2 Fold Increase P-Value Index Genes Log2 Fold Decrease P-Value 1 fam92b 11.14225 4.19E-16 1 pak3 -8.23733 7.22E-19 2 spag6 10.9692 2.27E-15 2 nos1 -8.04714 3.08E-14 3 hla-dra 10.76704 1.35E-55 3 fosb -7.83022 3.68E-262 4 olfm4 10.64141 4.83E-26 4 na -7.64523 1.16E-10 5 kit 10.61465 9.89E-22 5 glb1l3 -7.5987 9.38E-50 6 cfap221 10.57829 3.90E-15 6 shcbp1 -6.81588 1.66E-40 7 scgb3a1 10.22667 2.13E-113 7 na -6.81204 1.44E-08 8 bpifb1 10.22314 1.54E-143 8 slc8a1 -6.79175 2.85E-63 9 muc5b 10.2186 4.48E-103 9 ki67 -6.77092 8.46E-40 10 foxj1 10.21257 3.80E-18 10 rab3b -6.7198 3.31E-90 11 na 10.18837 2.81E-14 11 kif14 -6.71152 1.41E-47 12 c20orf85 10.17137 7.50E-19 12 dkk2 -6.7099 8.28E-13 13 ms4a8 10.13402 5.03E-12 13 na -6.70477 4.63E-09 14 fmo6p 10.0742 4.19E-21 14 pbk -6.67932 4.42E-39 15 foxi1 10.06812 7.53E-20 15 ckap2l -6.67208 1.09E-48

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Table 4.4.2- Up and down regulated genes in normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 20 versus day 0: Up- and down regulated genes out of 4,469 differentially expressed genes in NHBE-B day 20 versus NHBE-B day 0 ranked according to lowest adjusted p-value. Significant p-value ≤0.05. na=unidentifiable transcript sequences.

Up- and Down-regulated genes in NHBE-B 20 versus NHBE-B at Day 0

Index Genes Log2 Fold Increase Adjusted p-value 1 slc34a2 9.123016 <10E-300 2 pigr 9.36928 7.31E-291 3 fosb -7.83022 3.30E-258 4 tmprss4 5.701053 4.36E-230 5 ntn1 5.977488 4.51E-223 6 slc44a4 7.224355 2.38E-212 7 tjp3 7.24194 1.59E-210 8 loxl4 7.71815 2.20E-198 9 muc20 8.445317 3.76E-198 10 clic6 7.701496 3.30E-197 11 vtcn1 7.189049 1.89E-184 12 ceacam5 7.321699 4.83E-176 13 wfdc2 9.391115 7.42E-170 14 ppm1h 7.356917 8.03E-170 15 muc4 9.2582 2.26E-165

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Analysis of differentially activated pathways in NHBE-B at day 20

Pathways enhanced by 2-fold up- and down-regulated genes analysed in Reactome 2106 were those pertaining to metabolism particularly resembled the pathway profile seen at day 8 NHBE-B culture. Metabolic pathways such as fatty acid metabolism and oxidation pathways as well as protein glycosylation pathways were active. Down-regulated pathways included the cell cycle pathway and mitotic pathways (Table 4.4.3).

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Table 4.4.3-Pathway analysis of all the genes increased or decreased by ≥2-fold in normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) at day 20 compared to day 0: The top 10 pathways were enriched for the 3,301 up-regulated genes (shown in red) whilst the bottom 10 down-regulated pathways using 1,168 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR. na=unidentifiable transcript sequences. NHBE-B Day 20 vs Day 0 Pathway Analysis of Down-regulated NHBE-B Day 20 vs Day 0 Pathway Analysis of Up-regulated Genes Genes Adjusted Adjusted Index Name P-value Index Name P-value p-value p-value 1 Biological oxidations 4.24E-12 4.80E-09 1 Cell Cycle 2.34E-48 2.10E-45 Phase 1 - Functionalization of 2 5.00E-11 2.83E-08 2 Cell Cycle, Mitotic 1.57E-44 7.05E-42 compounds Defective GALNT12 causes colorectal 3 3.20E-09 9.05E-07 3 Mitotic Prometaphase 1.39E-27 4.17E-25 cancer 1 (CRCS1) Defective GALNT3 causes familial 4 hyperphosphatemia tumoral calcinosis 3.20E-09 9.05E-07 4 M Phase 5.92E-23 1.06E-20 (HFTC) Defective C1GALT1C1 causes Tn Resolution of Sister Chromatid 5 2.31E-08 3.74E-06 5 1.89E-23 4.24E-21 polyagglutination syndrome (TNPS) Cohesion Cytochrome P450 - arranged by Mitotic Metaphase and 6 6.97E-09 1.58E-06 6 9.80E-18 1.26E-15 substrate type Anaphase 7 Fatty acids 4.32E-08 6.11E-06 7 Mitotic Anaphase 5.16E-17 5.79E-15 8 Xenobiotics 2.31E-08 3.74E-06 8 Cell Cycle Checkpoints 3.23E-16 3.23E-14 RHO GTPases Activate 9 Termination of O-glycan biosynthesis 1.88E-07 2.36E-05 9 4.10E-18 6.14E-16 Formins Diseases associated with O- 10 7.76E-07 8.78E-05 10 RHO GTPase Effectors 7.83E-16 7.03E-14 glycosylation of proteins

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Analysis of differentially expressed genes in bronchospheres from COPD subjects at day 20

The master cilial transcription gene foxj1 was up-regulated 10-fold (log2) together with fam92b and dynein regulatory complex subunit 7 (drc7) which are both implicated in flagella and ciliary motility. Genes that participate in drug and oxidative stress metabolism including cytochrome P450 family 2 subfamily F Member 1 (cyp2f1) and fmo6p were also up-regulated at day 20 (Table 4.4.4).

Fosb and foxa2 genes that have crucial roles in cell differentiation and transformation were both significantly down-regulated in COPD bronchospheres at day 20 compared to day 0 (Table 4.4.4).

Genes involved in oxidative pathways such as arachidonate 5-lipoxygenase (alox5), dehydrogenase/reductase 9 (dhrs9), ornithine decarboxylase 1 (odc1), uridine phosphorylase 1 (upp1) and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (st6gal1) were significantly up-regulated when ranked according to adjusted p value (Table 4.4.5).

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Table 4.4.4- Top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) at day 20 and day 0 ranked according to log2 fold-change: The top 15 highly expressed transcripts shown in red from 3,244 significantly differentially expressed genes and the bottom 15 least expressed transcripts shown in blue out of 1,193 significantly repressed genes. na=unidentifiable transcript sequences.

CHBE-B Day 20 vs Day 0

Index Genes Log2 Fold Increase P-Value Index Genes Log2 Fold Decrease P-Value 1 fam92b 11.62052 3.09E-06 1 fosb -8.78711 1.25E-210 2 ms4a8 11.3497 9.15E-12 2 foxa2 -8.492 2.62E-129 3 c9orf135 11.14985 2.92E-09 3 pappa2 -7.94933 5.85E-47 4 drc7 10.9346 4.59E-05 4 linc02261 -7.50472 5.84E-12 5 fam216b 10.79329 2.11E-08 5 na -7.45184 3.71E-12 6 krt1 10.70081 4.05E-11 6 na -7.22125 1.67E-88 7 lrrc10b 10.68781 1.48E-08 7 trib3 -6.94182 5.76E-152 8 bpifa2 10.66909 3.07E-26 8 aldh1l2 -6.6983 5.87E-192 9 na 10.63389 2.72E-22 9 na -6.54107 2.02E-08 10 ankrd66 10.50885 3.45E-05 10 glb1l3 -6.50161 2.68E-117 11 cyp2f1 10.50156 2.03E-42 11 na -6.46621 3.65E-08 12 c20orf85 10.49451 8.15E-10 12 samd5 -6.46556 9.60E-22 13 fmo6p 10.41479 1.02E-22 13 cdh4 -6.46083 2.30E-101 14 olfm4 10.41046 6.63E-31 14 na -6.45836 2.57E-09 15 foxj1 10.37509 3.55E-17 15 na -6.44556 1.01E-08

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Table 4.4.5- The top 15 Up and down regulated genes in COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B) at day 20 versus day 0 ranked by adjusted p value: Up- and down regulated genes out of 4,437 differentially expressed genes in CHBE-B day 20 versus NHBE-B day 0 ranked according to lowest adjusted p-value. na=unidentifiable transcript sequences.

Up- and Down-regulated genes in COPD bronchospheres (CHBE-B) day 20 versus bronchospheres from normal healthy subjects (NHBE) at Day 0

Index Genes Log2 Fold Increase Adjusted p-value 1 vtcn1 5.491652 <10E-300 2 s100a4 4.484157 <10E-300 3 pigr 9.167499 <10E-300 4 odc1 -4.20533 <10E-300 5 dhrs9 6.902971 <10E-300 6 sidt1 4.104455 <10E-300 7 st6gal1 2.661002 <10E-300 8 gabrp 9.299891 <10E-300 9 adgrf1 4.180981 <10E-300 10 upp1 -4.56852 <10E-300 11 adam28 5.043221 <10E-300 12 rbpms 3.214219 <10E-300 13 rarres3 4.602082 <10E-300 14 vsig2 5.719615 <10E-300 15 alox5 6.306336 <10E-300

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Analysis of differentially activated pathways in CHBE-B at day 20

At day 20, metabolic pathways such as biological oxidation, fatty acid metabolism and drug metabolism (cytochrome 450 and functionalisation of compounds) and glycosylation pathways were significantly up-regulated in CHBE-B compared to day 0. ECM modification pathways were also significantly up-regulated (Table 4.4.6).

In contrast, the PI3K/AKT survival pathways were significantly down-regulated as were the FGFR1-4 pathways that are crucial for cellular differentiation and repair in the lung (Table 4.4.6).

Further direct comparisons of differentially expressed genes between CHBE-B and NHBE-B at day 20 should be undertaken along with an analysis of differentially expressed COPD development- and smoking-related pathways using gene set variation analysis (GSVA).

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Table 4.4.6-Pathway analysis of all the genes increased or decreased by ≥2-fold in COPD bronchial epithelial (CHBE) cell-derived bronchospheres (CHBE-B) day 20 compared to day 0: The top 10 pathways were enriched for the 3,244 up-regulated genes (shown in red) whilst the bottom 10 down- regulated pathways using 1,193 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR. na=unidentifiable transcript sequences. CHBE-B Day 20 vs Day 0 Pathway Analysis of Up-regulated Genes CHBE-B Day 20 vs Day 0 Pathway Analysis of Down-regulated Genes Adjusted Adjusted Index Name P-value Index Name P-value p-value p-value 1 Biological oxidations 3.85E-09 4.31E-06 1 PI3K/AKT activation 2.97E-06 0.001136 Constitutive Signalling by 2 Extracellular matrix organization 1.30E-07 7.29E-05 2 1.7E-06 0.001136 Aberrant PI3K in Cancer Negative regulation of the 3 Fatty acids 9.90E-07 0.00037 3 4.48E-06 0.001144 PI3K/AKT network Phase 1 - Functionalization of PI5P, PP2A and IER3 Regulate 4 1.81E-06 0.000507 4 9.06E-06 0.001734 compounds PI3K/AKT Signalling Defective GALNT12 causes 5 1.03E-05 0.001915 5 PI-3K cascade: FGFR1 3.81E-05 0.002646 colorectal cancer 1 (CRCS1) Defective GALNT3 causes familial 6 hyperphosphatemic tumoral 1.03E-05 0.001915 6 PI-3K cascade: FGFR2 3.81E-05 0.002646 calcinosis (HFTC) Cytochrome P450 - arranged by 7 2.03E-05 0.002846 7 PI-3K cascade: FGFR3 3.81E-05 0.002646 substrate type Defective C1GALT1C1 causes Tn 8 3.45E-05 0.004295 8 PI-3K cascade: FGFR4 3.81E-05 0.002646 polyagglutination syndrome (TNPS) Phosphorylation of CD3 and TCR 9 3.9E-05 0.004366 9 PI3K events in ERBB4 signalling 3.81E-05 0.002646 zeta chains 10 Collagen degradation 9.54E-05 0.007172 10 PIP3 activates AKT signalling 3.81E-05 0.002646

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4.5 Effect of cigarette smoke condensate (CSC) on gene expression profiles over time in normal healthy human bronchospheres (NHBE-B). The addition of CSC to NHBE for 2hrs resulted in fewer differentially expressed genes between CSC-NHBE-B and control NHBE-B culture (962 genes) than between CHBE vs NHBE cells (1,735) (Figure 4.5.1, Table 4.5.1 and 4.5.2).

Figure 4.5.1.-Baseline differential gene expression: Heat map showing the 30 top differentially expressed genes at Day 0 of CSC-treated normal healthy human bronchial epithelial (NHBE) cell bronchosphere culture (CSC-NHBE-B) compared with DMSO-treated NHBE-B. Overall, there were 962 differentially expressed genes between CSC-NHBE-B and control DMSO-treated bronchospheres (DMSO-NHBE-B) at day 0. 467 genes were up-regulated and 495 were down-regulated.

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Expression analysis of the top 15 out of 467 up-regulated genes included IL-36b (il36b), KIT protooncogene tyrosine kinase (kit), caspase 5 (casp5) and oncostatin M (osm) which are involved in cytokine and interleukin signalling and apoptosis (Table 4.5.1).

The top 15 out of 495 down-regulated genes included neuroglobin 4 Y-linked (nlgn4y), ribosomal protein S4 Y-linked 1 (rps4y1) ubiquitously transcribed tetraricopeptide containing Y-linked (uty). uty is associated with protein-protein interactions and particularly chromatin organisation.

Ranking the top 15 differentially expressed genes by adjusted p value highlights genes such as mmp1 associated with the breakdown of extracellular matrix and tissue remodelling.

CSC-NHBE-B had 2 up-regulated and 1 down-regulated transcripts compared to DMSO-treated NHBE-B that could not be identified indicating that there may be other transcript effectors that may be important in these pathways. Non-assigned transcripts were excluded from pathway analysis (Tables 4.5.1 and 4.5.2).

Due to time constraints, the further analysis of CSC-NHBE-B versus DMSO-NHBE-B at day 0 and day 20 have not been performed. A final analysis examining differentially expressed genes between NHBE-B, CHBE-B, CSC-NHBE-B and DMSO-NHBE-B should also be performed. Ongoing analysis on key COPD development and smoking pathways should also be undertaken using GSVA.

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Table 4.5.1- Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) and control NHBE-B at day 0 ranked according to log2 fold-change: Top 15 highly expressed transcripts shown in red from 467 significantly differentially up-regulated genes and the bottom 15 least expressed transcripts shown in blue out of 495 significantly repressed genes. na=unidentifiable transcript sequences.

CSC-NHBE-B Day 0 vs NHBE-B at Day 0

Index Genes Log2 Fold Increase Adjusted p-Value Index Genes Log2 Fold Decrease Adjusted p-Value 1 hmox1 7.166251 5.19E-07 1 nlgn4y -10.7753 2.30E-05 2 sv2b 6.947711 0.043567 2 uty -9.85498 0.000101 3 cnr1 6.705516 0.004401 3 zfy -9.35579 0.000169 4 cyp4f35p 6.663521 0.001519 4 eif1ay -8.8462 0.001293 5 mir3189 6.620994 0.001957 5 ddx3y -8.80229 9.32E-05 6 casp5 6.236059 0.024115 6 loc400655 -8.41241 0.001091 7 na 6.105261 0.034037 7 rps4y1 -8.12319 0.000112 8 slc6a12 6.082393 0.001301 8 prky -7.99944 0.000733 9 na 5.759292 0.004933 9 usp9y -7.95794 0.00033 10 igfbp1 5.636023 0.004642 10 ttty15 -7.75791 0.003601 11 kit 5.631188 0.020068 11 txlngy -7.26248 0.001097 12 il36b 5.624441 0.004268 12 na -7.1174 0.000732 13 osm 5.549551 0.022207 13 luaris -6.73696 0.002962 14 hgfac 5.46335 1.91E-05 14 gyg2p1 -6.6629 0.017707 15 na 5.448014 0.045075 15 na -6.6066 0.020506

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Table 4.5.2- Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) and control bronchospheres (NHBE-B) at day 0 ranked according to log2 fold-change: Up- and down regulated genes out of 962 differentially expressed genes in CSC-NHBE-B day 0 versus control NHBE-B at day 0 ranked according to lowest adjusted p-value. na=unidentifiable transcript sequences. na=unidentifiable transcript sequences.

Up- and Down-regulated genes in CSC-NHBE-B versus control NHBE-B at day 0

Index Genes Log2 Fold Increase Adjusted p-value 1 hspa6 4.468861 1.93E-08 2 raet1e -2.09782 2.10E-07 3 prss22 4.773831 2.10E-07 4 top2a -4.26915 5.19E-07 5 rhobtb2 -2.1337 5.19E-07 6 hmox1 7.166251 5.19E-07 7 Na 3.605701 1.32E-06 8 mmp1 3.550868 2.27E-06 9 depdc1 -3.86445 2.53E-06 10 cyp1a1 3.473771 3.26E-06 11 syn1 3.690467 3.26E-06 12 mig7 -2.79465 3.77E-06 13 cldn4 2.938167 3.94E-06 14 fa2h 3.218872 3.94E-06 15 tnfaip8l1 -3.27719 5.22E-06

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Analysis of differentially activated pathways in CSC-NHBE-B at day 0

Pathway analysis of ≥2 fold up-regulated genes showed the highest up- regulation in cell junction pathways such as those involved in tight junction formation. Interleukin signalling pathways were also up-regulated along with basigin signalling. Basigin is a cell surface glycoprotein that regulates signalling by MMPs such as MMP9 as well as interacting with ECM proteins such as integrins (Muramatsu, 2016, Jouneau et al., 2011) (Table 4.5.3).

Pathway analysis of genes that were ≤2-fold suppressed showed down regulation of cell cycle and cell division pathways including mitotic pathways (Table 4.5.3).

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Table 4.5.3-Pathway analysis of all the genes increased or decreased by ≥2-fold in cigarette smoke (CSC)-treated normal healthy human epithelial (NHBE)-derived bronchospheres (CSC-NHBE-B) at day 0 compared control NHBE-B at day 0: The top 10 pathways were enriched for the 663 up-regulated genes (shown in red) whilst the bottom 10 down-regulated pathways using 1072 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR. na=unidentifiable transcript sequences.

CSC-NHBE-B vs control NHBE-B at Day 0: CSC-NHBE-B vs control NHBE-B at Day 0: Pathway Analysis of Up-regulated Genes Pathway Analysis of Down-regulated Genes Index Name P-value Adjusted p-value Index Name P-value Adjusted p-value 1 Cell-cell junction organization 8.69E-05 0.04426 1 Cell Cycle 2.13E-27 1.38E-24 2 Tight junction interactions 0.000152 0.04426 2 Cell Cycle, Mitotic 1.45E-24 4.71E-22 3 Cell junction organization 0.00074 0.1433 3 Mitotic Prometaphase 7.92E-16 1.71E-13 Transmembrane transport of 4 0.001729 0.2009 4 M Phase 8.44E-13 1.09E-10 small molecules 5 LGI-ADAM interactions 0.001635 0.2009 5 Signalling by Rho GTPases 7.79E-13 1.09E-10 Resolution of Sister 6 Signalling by Interleukins 0.008906 0.5214 6 3.34E-11 3.61E-09 Chromatid Cohesion 7 Iron uptake and transport 0.006451 0.5214 7 Cell Cycle Checkpoints 2.40E-09 1.55E-07 8 Cell-Cell communication 0.007956 0.5214 8 RHO GTPase Effectors 1.45E-09 1.17E-07 Resolution of D-loop Structures through Synthesis- 9 Signalling by Insulin receptor 0.02046 0.5307 9 1.96E-09 1.41E-07 Dependent Strand Annealing (SDSA) 10 Basigin interactions 0.008974 0.5214 10 Mitotic G1-G1/S phases 5.42E-09 2.93E-07

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Analysis of differentially activated genes in CSC-NHBE-B at day 20

RNA-seq analysis of CSC-NHBE-B at day 20 of culture detected 1,884 differentially expressed genes compared with NHBE-B at day 0, which was fewer than between NHBE-B day 20 versus NHBE-B day 0 (4,469) and CHBE-B day 20 versus NHBE-B Day 0 (4,438).

Figure 4.5.2-Differential gene expression after 20 Days of bronchosphere culture: Heat map showing the 30 top differentially expressed genes at Day 20 of CSC-treated normal healthy human bronchial epithelial (NHBE) cell bronchosphere culture (CSC-NHBE-B) compared with NHBE-B at day 0. Chronic CSC exposure of NHBE-B resulted in 1884 differentially expressed genes at day 20 compared to day 0.

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The top 15 up-regulated genes at day 20 of CSC-NHBE-B culture included alcohol dehydrogenase 1C (Class I), gamma polypeptide (adh1c), arachidonate 15-lipoxygenase (alox15), cyp2f1, fmo2 and indoleamine 2,3-dioxygenase 1 (ido1) which are important in metabolic pathways particularly in oxidative stress. Mucin gene muc5b and goblet cell marker bpi fold containing family A member 2 (bpifa2 or splunc2) were also up-regulated (Table 4.5.4).

The top 15 down-regulated genes aldehyde dehydrogenase 1 family member L2 (aldh1l2) and apolipoprotein 1 (apoa1) which are important in vitamin metabolism which is important for cellular differentiation e.g. the metabolization of RA is important for cellular differentiation (Table 4.5.4).

Gene ranking by adjusted p-value revealed genes such as loxl4 and mmp13 which are involved in collagen degradation pathways. loxl4 and mmp13 feature in ECM reorganisation pathways with ceacam6 and capn13. Mucin genes muc5b and muc4 were also up-regulated and are part of O-linked glycosylation pathway (Table 4.5.5).

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Table 4.5.4- Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) at day 20 and control bronchospheres NHBE-B at day 0 ranked according to log2 fold-change: Top 15 highly expressed transcripts shown in red out of 1,449 up-regulated genes and the bottom 15 least expressed transcripts of shown in blue of 435 down- regulated genes after CSC treatment of NHBE-B on day 20 versus NHBE-B at day 0. na=unidentifiable transcript sequences.

CSC-NHBE-B at Day 20 versus HBE-B at Day 0

Index Genes Log2 Fold Increase Adjusted p-Value Index Genes Log2 Fold Decrease Adjusted p-Value 1 na 9.501229 8.85E-08 1 ntsr1 -9.96488 2.30E-06 2 cdc20b 9.49457 6.58E-13 2 krt75 -8.89205 1.09E-11 3 cdhr4 9.467452 2.53E-10 3 na -8.58234 0.000623 4 cfap74 9.418652 1.28E-07 4 glb1l3 -8.15134 1.77E-20 5 ldlrad1 9.415124 1.38E-12 5 il13ra2 -8.04037 7.18E-24 6 muc5b 9.379015 1.30E-22 6 aldh1l2 -7.93389 2.09E-16 7 ido1 9.317796 1.51E-10 7 na -7.49539 2.34E-06 8 fmo2 9.28415 1.67E-13 8 na -7.49184 0.030725 9 cyp2f1 9.209308 2.48E-12 9 tmem119 -7.49184 0.030725 10 c9orf135 9.181813 1.88E-07 10 ca7 -7.49184 0.030725 11 adh1c 9.176381 3.06E-10 11 na -7.44065 0.004634 12 bpifa2 9.093245 1.84E-10 12 na -7.41527 0.000401 13 ankrd66 9.04994 5.79E-07 13 msx1 -7.37519 1.14E-05 14 alox15 9.033527 1.06E-16 14 kcnk3 -7.08802 0.035934 15 lrrc10b 8.994654 8.33E-10 15 apoa1 -7.0545 0.044859

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Table 4.5.5- Top differentially expressed genes between cigarette smoke condensate (CSC)-treated normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (CSC-NHBE-B) at day 20 and control bronchospheres (NHBE) at day 0 ranked according to log2 fold-change: Up- and down regulated genes out of 1,884 differentially expressed genes in CSC-NHBE-B day 20 versus NHBE-B day 0 ranked according to lowest adjusted p-value. na=unidentifiable transcript sequences.

Up- and Down-regulated genes in CSC-NHBE-B at Day 20 versus NHBE-B at Day 0

Index Genes Log2 Fold Increase Adjusted p-value 1 muc4 7.711027 3.22E-27 2 bpifb1 8.072146 3.22E-27 3 cp 7.95836 1.25E-26 4 il13ra2 -8.04037 7.18E-24 5 ceacam5 8.002785 9.03E-24 6 ceacam6 7.108898 3.80E-23 7 gabrp 7.944518 4.26E-23 8 pigr 7.652298 4.26E-23 9 loxl4 7.027151 4.26E-23 10 krt4 7.223072 5.67E-23 11 muc5b 9.379015 1.30E-22 12 mmp13 7.50777 3.13E-22 13 tf 8.838633 9.73E-22 14 capn13 8.24336 1.96E-21 15 cyp2b7p 7.815804 2.29E-21

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Analysis of differentially activated pathways in CSC-NHBE-B at day 20

Pathway analysis of genes by ≥2-fold expression showed up-regulation of oxidative pathways as well as of protein O-linked oligosaccharide biosynthesis pathways like those observed in CHBE-B (Table 4.5.6).

In addition, pathway analysis of genes expressing ≤-2-fold showed down-regulation of cell survival pathways as well as pathways signalling though FGFR1-4 receptors and PI3K as observed earlier with CHBE-B at day 20 (Table 4.5.6).

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Table 4.5.6- Pathway analysis of all the genes increased or decreased by ≥2-fold in cigarette smoke (CSC)-treated normal healthy human epithelial (NHBE)-derived bronchospheres (CSC-NHBE-B) at day 20 compared control NHBE-B at day 0: The top 10 pathways were enriched for the 1,449 up-regulated genes (shown in red) whilst the bottom 10 down-regulated pathways using 435 genes are indicated in blue. Pathway analysis was performed using the Reactome pathway database within EnrichR. na=unidentifiable transcript sequences.

CSC-NHBE-B Day 20 vs Day 0: CSC-NHBE-B Day 20 vs Day 0 Pathway Analysis of Up-regulated Genes Pathway Analysis of Down-regulated Genes Adjusted Adjusted Index Name P-value Index Name P-value p-value p-value Defective GALNT12 causes Amino acid transport across the 1 5.66E-07 0.000259 1 2.2E-05 0.01484 colorectal cancer 1 (CRCS1) plasma membrane Defective GALNT3 causes familial Negative regulation of the 2 hyperphosphatemic tumoral 5.66E-07 0.000259 2 5.24E-05 0.0177 PI3K/AKT network calcinosis (HFTC) Termination of O-glycan 3 2.61E-06 0.000477 3 PI3K/AKT activation 0.000136 0.02388 biosynthesis Defective C1GALT1C1 causes Tn PI5P, PP2A and IER3 Regulate 4 polyagglutination syndrome 1.74E-06 0.000397 4 0.000177 0.02388 PI3K/AKT Signalling (TNPS) Phase 1 - Functionalization of Constitutive Signalling by 5 1.41E-06 0.000397 5 0.00015 0.02388 compounds Aberrant PI3K in Cancer 6 O-linked glycosylation of mucins 3.51E-06 0.000535 6 PI-3K cascade: FGFR1 0.000533 0.03 7 Biological oxidations 4.97E-06 0.00065 7 PI-3K cascade: FGFR2 0.000533 0.03 Amino acid and oligopeptide 8 O-linked glycosylation 2.43E-05 0.002784 8 0.000313 0.03 SLC transporters Diseases associated with O- 9 0.000143 0.01308 9 PI-3K cascade: FGFR3 0.000533 0.03 glycosylation of proteins 10 Interferon gamma signalling 9.03E-05 0.009194 10 PI-3K cascade: FGFR4 0.000533 0.03

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Comparison of differentially expressed genes across groups

Out of the 15 highest expressed transcripts, no transcripts were expressed by all 3 conditions at day 20 of culture. 6 transcripts: chromosome 20 open reading frame 85 (c20orf85), fam92b, fmo6p, foxj1 and membrane spanning 4-domains A8 (ms4a8) and olfactomedin 4 (olfm4) were expressed by both NHBE-B and CHBE-B (Table 4.5.7). Only 1 transcript, muc5b, was expressed by both NHBE-B and CSC-NHBE-B whereas 5 transcripts, cyp2f1, chromosome 9 open reading frame 135 (c9orf135), bpifa2 (splunc2), ankyrin repeat domain 66 (ankrd66) and leucine rich repeat coding 10B (lrrc10b) were highly expressed by both CHBE-B and CSC-NHBE-B at day 20 of bronchosphere culture (Table 4.5.7).

Out of the bottom 15 lowest expressed transcripts, all conditions showed a low expression in galactosidase beta 1 like 3 (glb1l3) transcript. NHBE-B and CHBE-B both down-regulated expression of fosb whereas no commonly down-regulated transcripts were observed between NHBE-B, CSC-NHBE-B and CHBE-B (Table 4.5.7). However, pathways such as PI3K were down-regulated in both CSC-NHBE-B and CHBE-B at day 20.

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Table 4.5.7- Comparison of top differentially expressed genes between COPD bronchial epithelial (CHBE)-derived bronchospheres (CHBE-B), normal healthy human bronchial epithelial (NHBE) cell-derived bronchospheres (NHBE-B) and cigarette smoke condensate (CSC)-treated NHBE-B (CSC-NHBE-B) ranked according to log2 fold-change at day 20 compared to day 0: The top 15 highly expressed transcripts for NHBE-B (3,301 genes), CSC-NHBE (1,449 genes) and CHBE-B (3,244 genes) shown in red and the bottom 15 least expressed transcripts for NHBE-B (1,168 genes), CSC-NHBE (435 genes) and CHBE-B (1,193 genes) shown in blue. na=unidentifiable transcript sequences.

Up-regulated genes Down-regulated genes Index NHBE CSC-NHBE CHBE Index NHBE CSC-NHBE CHBE 1 fam92b na fam92b 1 pak3 ntsr1 fosb 2 spag6 cdc20b ms4a8 2 nos1 krt75 foxa2 3 hla-dra cdhr4 c9orf135 3 fosb na pappa2 4 olfm4 cfap74[201] drc7 4 na glb1l3 linc02261 5 kit ldlrad1 fam216b 5 glb1l3 il13ra2 na 6 cfap221 muc5b krt1 6 shcbp1 aldh1l2 na 7 scgb3a1 ido1 lrrc10b 7 na na trib3 8 bpifb1 fmo2 bpifa2 (splunc2) 8 slc8a1 na aldh1l2 9 muc5b cyp2f1 na 9 ki67 tmem119 na 10 foxj1 c9orf135 ankrd66 10 rab3b ca7 glb1l3 11 na adh1c cyp2f1 11 kif14 na na 12 c20orf85 bpifa2 (splunc2) c20orf85 12 dkk2 na samd5 13 ms4a8 ankrd66 fmo6p 13 na msx1 cdh4 14 fmo6p alox15 olfm4 14 pbk kcnk3 na 15 foxi1 lrrc10b foxj1 15 ckap2l apoa1 na

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4.6 Discussion RNA-seq analysis revealed that at baseline (post basal cell resuspension in matrigel for 2 hours), NHBE, CSC-NHBE and CHBE cells already showed differential gene expression (Figure 4.2.1 and 4.5.1). Basal cells from the different disease categories at day 0 had different transcript profiles that may indicate they have become inherently different.

NHBE-B, CHBE-B and CSC-NHBE-B from day 0-20 up-regulate biological oxidation pathways to regulate oxidative stress (Tables 4.2.3, 4.3.3, 4.3.6, 4.4.3, 4.4.6, 4.5.3 and 4.5.6) and therefore must be a common trait of the bronchosphere culture. This may be due to the culture mimicking oxidative stress during development. Although hypoxic environments have been shown to cause disease and are one of the major features in the adult lung, hypoxia is essential for organogenesis and differentiation of cells in foetal lungs. Foetal development occurs in a mainly hypoxic environment in the first trimester in utero (van Tuyl et al., 2005, Gebb and Jones, 2003, Webster and Abela, 2007). The resulting oxidative stress triggers cell migration, proliferation, differentiation, angiogenesis. Upon uteroplacental circulation establishment, antioxidant defences are enhanced leading to a drop in oxidative stress (van Tuyl et al., 2005, Gebb and Jones, 2003, Gebb et al., 2005). The drop in ROS from oxidative stress and increase in nutrition triggers cellular differentiation and causes a reduction in proliferation (Castagne et al., 1999, Han et al., 2018).

Figure 4.6.1- Response of cells to REDOX switching: Blue-red indicates increasing oxygen levels.

The change of cell behaviours in response to oxidative stress is known as REDOX switching (Figure 4.6.1). Therefore, oxidative and antioxidative pathways are in a delicate balance and their perturbation may lead to incorrect cell fate. These observations could apply to the bronchosphere model as cell cycle and mitotic pathways were down regulated in NHBE-B at day 8 of culture where IF staining

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showed NHBE-B begin to form a lumen. A less proliferative phenotype could be due to oxygen tension as the central spheroid population in the growing bronchosphere would not receive as much oxygen as the outer population. This may be a trigger for cell polarisation and differentiation.

However, CHBE-B and the treatment of NHBE with CSC at day 0 showed a down regulation in cell cycle and proliferation (Table 4.2.3 and 4.5.3). CHBE-B had delayed differentiation and luminal formation whilst the lumen did not develop in CSC-NHBE-B even by day 20 (Chapter 3, Figures 3.2.4, 3.2.5, 3.3.2 and 3.3.3). Cell cycle down-regulation in this context may be causal in the delay in luminal formation and differentiation. CSC is known to stall the cell cycle and cellular senescence has previously been observed in COPD and smoker patients (Zhao et al., 2009, Zhou et al., 2011). Further investigation will be required to determine the correlation between the cell cycle and cellular differentiation.

At days 8 and 20 of CHBE-B culture, ECM reorganisation as well as ECM degradation and collagen degradation pathways were up-regulated (Table 4.3.6 and 4.4.6). A central characteristic of COPD is excess matrix degradation (due to up-regulation of MMP proteins such as mmp9) and deposition that cause the airway wall to undergo structural changes in ECM and stiffen (Yao et al., 2010, Sand et al., 2015, Sand et al., 2016). Oxidative stress in COPD has been linked to these features in several studies (Gao et al., 2008, Kliment et al., 2008, Lee et al., 2007). NHBE-B did not show an up-regulation in matrix degradation at any of the time-points studied thus reflecting airway biology.

The cilia associated gene fam92b and the cilia master transcription factor, FOXJ1 (Chapter 3) were up-regulated in both NHBE-B and CHBE-B ant day 20. However, another 2 highly expressed transcripts, cilia and flagella associate protein 221 (cfap221) also known as primary cilial dyskinesia protein 1 (pcd1)) and spag6 were not up-regulated in CHBE-B (Table 4.5.7).

CFAP221 protein is found in the cilia of brain epididymis, inner ear, upper and lower respiratory epithelial cells as well as flagella of eukaryotic cells such as sperm (Lee et al., 2008). CFAP221 deletion from mice results in complete loss of sperm flagella and 25% reduction in respiratory cilial beat frequency although ultrastructure

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analysis of cilia show them as being normal (McKenzie et al., 2013, Lee et al., 2008). Transgenic restoration of CFAP221 rescues the phenotype (Teves et al., 2014, Lee et al., 2008). It is possible that CFAP221 is involved in the regulation of the dynein motor and not with cilial microtubule extension as mice with CFAP211 deletions have a similar phenotype to mice with deletions in other dynein genes. CFAP221 deletion in mice also results in hyphocephalus, infertility and enhanced susceptibility to infection (Lee et al., 2008).

SPAG6 is found in cilia and flagella and have a well-documented role in regulating cilial length and motility. Its deletion results in infertility and disrupted ciliary beat frequency (Li et al., 2015, Sapiro et al., 2002). However, SPAG6 protein function is not just limited to cilia/flagella but is important in cell polarisation. spag6-/- mice die from hypocephalus although ciliary 9+2 axonemes in neuronal and tracheal are intact (Teves et al., 2014). Recent investigation in the brain, ependymal, trachea and middle ear epithelial cells has revealed that dysfunction in the axoneme/basal feet orientation due to disordered distribution microtubules interferes with normal cilia/basal polarity-dependent cellular polarisation (Teves et al., 2014). Therefore, spag6 expression has a key role in ciliogenesis and cellular polarity and its dysregulation causes severe disease related to cilial dysfunction.

One highly expressed cilium associated transcript drc7, was seen in CHBE-B but was not found in the top 15 highly expressed transcripts of NHBE-B day 20 (Table 4.5.7). The role of DRC7 is poorly understood, and it is predicted to be in the axoneme and regulate motility. Mutations and deletions in other DRC proteins such as DRC1, 2 and 4 have shown are found in several ciliopathies (Lin et al., 2011, Wirschell et al., 2013, Jeanson et al., 2016, Bower et al., 2018).

Only cfap74 (Table 4.5.4 and 4.5.7), a cilial marker found in cilia/flagella, was up-regulated in CSC-treated NHBE-B. In contrast, up-regulation of other cilia transcripts, particularly foxj1, were observed at day 20 in both NHBE-B and CHBE-B. The delayed increase in ciliary genes is in keeping with phenotypic development of CSC-NHBE-B seen in chapter 3 and a prolonged time-course of CSC-NHBE-B culture may reveal the expression of further dysregulated ciliary genes.

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The absence of expression of cfap221 and spag6 may be a reason for the reduced ciliary beat frequency of CHBE-B spheres and the delayed luminal formation since they both have important roles in ciliogenesis and apical basal polarisation of respiratory cells. These genes have not been investigated with respect to COPD and merit further study.

The presence of muc5b, scgb1a1 (not shown but the 20th most highly expressed transcript) and scgb1a3 at day 20 of NHBE-B culture may indicate the presence of club cells in the culture (Table 4.4.1). Club cells are only found in bronchioles of the conducting airways and therefore if present would suggest that bronchospheres in the study were representative of the lower airways. Club cells provides protective function through immune system modulation, secretion of anti-inflammatory proteins, oxidative stress reduction and xenobiotic processing. Club cells have also been shown to function as a progenitor population for ciliated cells differentiating to replenish ciliated cells (Rawlins et al., 2009b, McCauley et al., 2018). Analysis of epithelial subtypes including Club cells by gene set variation analysis (GSVA) may provide further insight into the model.

The scgb1a1 transcript was highly expressed at day 8 but was lost by day 20 of CHBE-B culture (Table 4.3.4 and 4.4.4). The club cell population is reduced in airways of smoker and COPD patients (Gamez et al., 2015). The reduction of the club cell population has been shown to result in increased inflammation, oxidative stress, squamous metaplasia and impaired airway repair observed in COPD (Laucho- Contreras et al., 2018).

foxa2 transcript expression was amongst the 15 lowest for CHBE-B at day 8 and day 20 culture (Table 4.3.4 and 4.4.4). Deletion of foxa2 arrests lung development in neonatal mice. A reduction of foxa2 in the distal conducting airways of mice results in a decrease in ccsp mRNA expression and an increase in goblet cell markers such as muc5ac (Wan et al., 2004). Furthermore, an inflammatory profile similar to that seen in COPD and other lung diseases is acquired in mice and human studies where goblet cells lack FOXA2 staining (Perl et al., 2002a, Perl et al., 2002b). However, other factors probably contribute to goblet cell hyperplasia as not all cells that have reduced foxa2 expression become goblet cells. The mechanism for the loss of club cells in COPD is

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not clear. Therefore, the bronchosphere model may be a better platform to study COPD not only for the development of different lineages and epithelial remodelling, but because bronchospheres may be modelling the small airways where the obstruction in COPD occurs overcoming the limitations of current models.

muc5b was also expressed by CSC-NHBE-B at day 20 of culture, but no other secretoglobin expression was observed. The day 20 CSC-N-HBE-B transcriptomic profile of the most highly expressed transcripts was more akin to that observed in CHBE-B than in NHBE-B with 5 transcripts in common. The transcript overlap between CSC-N-HBE-B and CHBE-B and the lack of overlap between NHBE-B may indicate a shift away from the NHBE-B phenotype to a diseased CHBE-B phenotype (Table 4.5.7). (Table 4.4.6 and 4.5.7) Furthermore, CSC-NHBE-B and CHBE-B had 8 similar down-regulated pathways at day 20. All down-regulated pathways involved PI3K signalling mostly through FGFR1-FGFR4 receptors (Table 4.4.5 and 4.5.6).

FGFR 1, 2 and 4 are expressed in large and lower airways whilst alveolar cells express FGFR2, 3, and 4. FGFRs, and FGFR2 in particular, are crucial for correct lung development and homeostasis and their dysregulation is associated with diseases such as IPF and cancer (MacKenzie et al., 2015, Ornitz and Itoh, 2015).

The role of FGFRs in COPD development and progression is poorly understood and there is a lack of studies in this area. However, as previously described (Introduction section 1.2.2) the role of FGFs and FGFRs in lung development and repair is well documented (Ornitz and Itoh, 2015).

A recent study showed that the FGFR1 protein was enhanced in the epithelium and ASM in resected lung sections of COPD patients as well as in patients with IPF (Kranenburg et al., 2005, MacKenzie et al., 2015). Another study showed that FGFR2 protein is enriched in lung epithelial cells but is down-regulated in CSC-treated healthy and COPD cells. Interestingly, decreased FGFR2 levels have been also been observed in regions with active airway remodelling in murine IPF lungs (El Agha et al., 2018). Clearly there is a need for a more in-depth mechanistic study to elucidate the exact mechanism of FGFs and FGFRs in development and regulation of COPD and the effect of cigarette smoke.

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At day 20, pathways in NHBE-B, CSC-NHBE-B and CHBE-B were enriched for protein O-glycosylation (Table 4.4.3, 4.4.6 and 4.5.3). O-glycosylation in development is necessary for cell surface presentation of proteins for cell-cell and cell-ECM adhesion, apical-basal polarity (important for bronchosphere lumen generation) and differentiation. O-glycosylation also is crucial for secretion of ECM, ECM composition and subsequent ECM signalling. These features seen during NHBE-B culture correlate with the differentiating, proliferating, ECM restructuring, cell-cell and cell-ECM adhesion changes observed in development (Vij and Zeitlin, 2006, Tian et al., 2012, Zhang and Ten Hagen, 2010, Zhang et al., 2010). O-glycosylation pathways may be necessary for the maintenance of the NHBE-B phenotype. It would be interesting to further explore the O-glycosylation status of CHBE-B and the effect CSC has on NHBE-B O-glycosylation with respect to the delayed effect on ECM and on differentiation.

NHBE-B, CSC-NHBE-B and CHBE-B transcriptional profiles display many of the hallmarks seen in never smoker, smoker and COPD donors. Therefore, further confirmation of up and down regulated transcripts through QPCR, gene knock down and protein and secreted protein analysis through Western blotting and enzyme linked immunosorbent assay (ELISA) are required to confirm transcript profiles to thoroughly characterise this model.

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5 Chapter 5

Effect of Epithelial-Stromal Cell Interaction in Bronchosphere Culture

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5.1 Epithelial-Stromal Interaction in 3 Dimensions The previous chapters aimed to define the role of epithelial cells in COPD. However, as discussed previously, the lung epithelium does not exist independently of other cell populations within the airway wall. Epithelial-stromal crosstalk is important in the paterning, maintenance and regeneration of the lung.

Epithelial-stromal crosstalk during development is necessarry for lung branching morphogenesis and the differentiation of progenitor cell populations into various linneages (Shannon et al., 1998, Alanis et al., 2014, El Agha et al., 2014). Once lung development is concluded, epithelial-stromal communication does not end. Crosstalk allows inactivation of developmental genes such as fgf10 to maintain lung structure by ensuring cell populations stay in the correct location (Nyeng et al., 2008). Upon injury, these mechanisms are once again activated to elicit repair. For example epithelial cells secrete WNT7b that signals ASM cells to secrete FGF10, which subsequently activates FGFR2b on basal epithelial cells, signalling cellular expansion at site of luminal injury (Volckaert et al., 2017). Crosstalk between the epithelium and fibroblast cells in the ECM is necessary for the reconstruction of the ECM as well as mediating the levels of basal epithelial cellular proliferation at the site of injury through TGFβ and EGRF signalling (Nishioka et al., 2015, Zhang et al., 1999, Royce et al., 2009).

Epithelial and stromal cells are dysregulated in COPD. Several studies have shown that stromal cell dysregulation may play a major role in ECM thickening, epithelial remodelling, tissue degredation and inflammation due to the abnormal repair processes ellicited in response to continuous lung injury in chronic pulmonary diseases such as asthma, COPD and IPF (Shaykhiev et al., 2011, Prasad et al., 2014, Nishioka et al., 2015). In COPD, goblet cell hyperplasia, ciliated cell hypoplasia and basal cell metaplasia are commonly observed and are reviewed extensively elsewhere (Randell, 2006).

The complexity of airway structure has made it difficult to model cell-cell communication, and the absence of adequate models has resulted in lack of research. Models that aim to imitate cell-cell communication usually involve cultures where epithelial and fibroblast or ASM monolayers are separated by a semi-permiable membrane (Shi et al., 2015, Prasad et al., 2014, Hsieh et al., 2005, Malavia et al.,

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2009, Hackett et al., 2011). Seperation of epithelial and stromal layers such as ASM and NHLF cells may result in changes to signalling due to the lack of physical interaction between cell layers (Holgate et al., 2004). Epithelial, NHLF and ASM layers are in constant comunication in respose to environmental stimuli. For example NHLF secrete the ECM that give cues for basal epithelial cell differentiation (Dupont et al., 2011). NHLF cells can also be altered into a more stiffer myofibroblast phenotype by their proximity to ASM which can alter ECM secreted (Hinz, 2007). The cells also signal to the immune system, but this area of crosstalk is beyond the scope of this thesis. As previously discussed, the ECM has a major role in organ homeostasis and cell-cell signalling. The absence of ECM in monolayer cultures may influence cellular signalling and behaviour that make it difficult to identify perturbed mechanisms and therefore drug targets for therapy in chronic diseases lung such as COPD.

A B

Figure 5.0.0 Airway wall structure: A- airway ciliated and goblet cells form a barrier that faces the lumen and block noxious elements in the air from entering the lung wall, B- Fibroblasts cells provide structure by secreting extracellular matrix (ECM) proteins that interact to form an elastic ECM layer. The basement membrane (BM) anchors epithelial cells to the lung wall. Fibroblast cells are active during repair of the epithelium restoring ECM signalling to inflammatory factors, C- Airway smooth muscle (ASM) dilates and constricts the airway to alter the airway lumen. ASM and epithelial crosstalk inhibits proteins such as fibroblast growth factor 10 (FGF10) to maintain a quiescent cell population.

This chapter will aim to model the effect of stromal cells on basal epithelial cells in bronchosphere culture.

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5.2 NHBE-NHLF co-culture results in tubular branching organoids In the first preliminary experiments, NHBE and NHLF were seeded into wells as described in the Methods Chapter (Methods, Section 2.2). At concentrations of NHLF >150,000 cells/ml epithelial cells aggregated as described previously in Chapter 3. However, above this concentration, aggregated spheres migrated under the gel and began to grow rod like structures (Figure 5.1.0-5.1.2) connecting to other spheres. At 450,000 - 1,350,000 cell/ml this process occurred more rapidly and discreet rods of 10-20μM were observed within 24hrs that merged together to form 50-70μM lumens by Day 3 (Figure 5.1.1-5.1.3) that were termed bronchotubules. These structures were short lived and collapsed from Day 4-6. The culture after 4 days became technically demanding as the media turned yellow within 12 hours and more frequent feeding was required. This indicates a decrease in pH reflecting enhanced growth, nutrient reduction and excessive waste product generation. Interestingly, growing tubule tips had rounded, bulbous tips as observed during branching morphogenesis in mice foetuses (Figure 5.1.2 D). Tube lumens showed no evidence of differentiation as cilia were not observed. Control NHBE spheroids also failed to show luminal differentiation at 4 days as previously shown in Chapter 3.

However not all epithelial cells formed tubular structures as those spheroids that had not migrated into the gel and remained on the surface stayed as spheroids with lumens (Figure 5.1.4).

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Figure 5.1.1- Normal healthy human bronchial epithelial (NHBE) cell and normal healthy human lung fibroblast (NHLF) co-culture: NHLF seeding density was increased from 6,000-1,350,000 cells/ml and NHLF cells allowed to adhere to the bottom of wells of a 96 well plate before being layered with 25% matrigel and then seeding NHBE cells in 5% matrigel. Increases in NHLF resulted in increased numbers of tubular structures formed by epithelial cells. Images are representative of those from 2 wells from each experiment and n=3 biological repeats. 10x magnification Scalebar = 200 μM

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Figure 5.1.2-Bronchotubule formation: Normal healthy human bronchial epithelial (NHBE) cells first aggregated into spheroids (A) that grew long rod like protrusions (B) that eventually formed into an interconnected set of tubular structures that looked ganglionic (C and E). Extending tubules proximally formed a bulbous structure that branched (D). Images are representative of those seen with n=3 donors. Scalebar=100 μM

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Figure 5.1.3-Bronchotubule morphology: Rod like structures formed lumens by Day 3 (red arrows A-C). Results are representative of bronchotubule formation in 2 wells per plate and in n=3 biological repeats. Scalebar = 100 μM

Figure 5.1.4-Cell behaviour above gel: Epithelial cells that did not migrate into the gel formed into spheroids and did not form rod like or tubular structures. Images are representative of those seen in 2 wells per plate and for n=3 donors. Scalebar = 100 μM.

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5.3 NHBE-NHLF-NHASM Triple Culture To supply enough media to the culture, transwells were used and cells were fed apically and basally. To prevent the collapse of tubules observed in Section 5.1.0 and to mimic morphology of airways observed in vivo, NHASM were added to the culture. The stromal cell concentrations were kept at 1,350,000 cells/ml (33,750 cells each for NHLF and NHASM). To investigate the effect of NHBE concentration on bronchotubule formation, NHBE cell numbers were varied (Figure 5.2.1).

Figure 5.2.1-NHBE concentration optimisation: NHBE concentration was varied while NHLF and NHASM seeding density was kept constant at 675,000 cells/ml. 10x magnification, scalebar = 100 μm. Images are representative of those from n=3 donors. The addition of NHASM did not stabilise bronchotubules, which collapsed after 4 days as observed in the previous single cell protocol used in Section 5.1.0. However, preliminary investigations showed that tubules contracted in response to the muscarinic receptor agonist carbachol (10-3M). In one instance tubules were also observed contracting independently of the addition of an antagonist (Figure 5.2.2).

Co-culture of NHLF+NHASM cells in the absence of NHBEs did not result in the formation of rods or tubules. Increasing the numbers of NHBE cells increased the size of the tubules obtained. No cilia were observed in lumens of tubules. The branching was not fractal and the ordered growth observed in the lungs in vivo was not seen but was more comparable to the morphology of a salivary gland (Patel and Hoffman, 2014). Tubules formed from NHBE (450,000 cells/ml), NHLF and NHASM cell (both at 675,000 seeding density) grew in a controlled manner resulting in the thickest 180

tubules (~200-500 μm) and therefore these seeding densities were used in subsequent experiments (Figure 5.2.1). A

C 150

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120 Area

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HBSS 10^-3 Carbocol Mol

Figure 5.2.2-Normal healthy human airway smooth muscle (HASM) Contraction assay: The functionality of NHASM was measured by the addition of the muscarinic against carbachol (10-3M) which causes ASM to contract and airway constriction compared to hank’s balanced salt solution (HBSS) buffer control. Purple bar shows luminal contraction, A, area of lumen was measured using imageJ by marking the inside lumen (blue and red), B and C. 40x magnification, Scale bar scalebar= 100 μm. n=1

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5.4 Scaffold Optimisation Since the addition of NHASM did not prevent tubule collapse, the scaffold stiffness was investigated. 25% matrigel was stiffened by adding the bio-unreactive seaweed polymer agarose to form an agarose-matrigel composite termed agrigel. Rheometry was used to measure the viscoelasticity of the agrigel (Figure 5.3.1). In dynamic mechanics, the storage modulus (G’) measures the stored energy in a material representing the elastic portion and the loss modulus (G”) is the energy lost by heat representing the viscous phase. A material is viscoelastic when G’ dominates G”. Further information on rheology of viscoelasticity can be found here (Callies et al., 2017). Agarose was assessed alone to determine its rheological properties. Increasing concentrations of agarose resulted in ~10-fold increase for G’ and G” from 0.3-1.0% agarose w/v suggesting that increasing the agarose concentration increases the stiffness of the gel (Figure 5.3.1).

1000A 10B

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Figure 5.3.1-Viscoelastic properties of agarose gel scaffold: (A) Storage modulus (G’) and loss of modulus G” (B) of agarose at different concentrations. (C) Storage modulus of agarose at different concentrations measured at 1Hz. (D) Loss modulus of agarose at different concentrations at 1Hz. Results are presented as mean±SEM of n=3 independent experiments.

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25% matrigel behaved as an elastic gel where G’ dominates G”. G’ and G” increased with increased agarose concentrations. The G’ of the highest concentration of agarose investigated (1.0%/25% agrigel) plateaued at 25Pa. Compared to agarose only, the agrigel G’ increased between 10-100-fold at lower oscillations, whilst minimal fold-increases were observed for G”.

Cells do not oscillate above 1Hz and therefore the data was analysed at 1Hz. Whilst G’ and G” of 0.3%/25% agrigel was like 25% matrigel, G’ of the 0.5%/25%- 1.0%/25% agrigel combinations were significantly higher (~3-5 fold) than that of 25% matrigel and 0.3%/25% agrigel. However, increasing the agarose concentration only resulted in a small increase in G’ in 0.3%-1.0% agarose only samples. The G” of agrigel increased by ~10-1000-fold with increasing concentration of agarose for all agrigel samples whereas a maximal ~10-fold increase was observed with agarose only samples (Figures 5.3.1-5.3.2).

These data showed that gel compliance decreased whilst gel elasticity was maintained in agrigel compared to 25% matrigel. Agrigel was more elastic and less compliant than agarose alone. The reduction of matrigel compliance is further demonstrated in Figure 5.3.2 Ei-Eii where 1.0%/25% matrigel can hold its shape whereas 25% matrigel alone is a droplet because it is a brittle material.

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A B 1000 10

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Figure 5.3.2- Viscoelastic properties of agrigel scaffold: (A) Storage modulus of agarose at different concentrations. (B) Loss modulus of agarose at different concentrations. (C) Storage modulus of agarose at different concentrations measured at 1Hz. (D) Loss modulus of agarose at different concentrations measured at 1Hz. (E(i)) Clarity of agrigel scaffold blue dot below gel can be clearly visualised. (E(ii)) Stiffness of agrigel increases with increasing agarose concentration. All data is presented as mean ± SEM of n=3 independent experiments using ANNOVA compared to 25% matrigel. *p<0.05, **p<0.01, ***p<0.001. For cell culture, 0.3%/25% and 1.0%/25% agrigel combinations were discounted. Either the agarose solidified before the gels could be mixed, the resultant gel formed

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a solid agarose gel surrounded by matrigel (0.3%/25%) or formed disparate aggregates of matrigel surrounded by agarose gel (1.0%/25%). Therefore, 0.5%/25% and 0.7%/25% agrigel scaffolds were analysed further (Figures 5.3.2-5.3.4).

The optimal properties of the scaffold required for tubule formation included the scaffold needing to be viscous enough to allow cellular migration for cellular aggregation at lower oscillations (<1Hz). However, the scaffold also must be stiff enough at higher oscillations to support larger bronchotubule structures.

At lower oscillations 0.7%/25% agrigel displayed a rubber phase where G’ and G” plateaued, whereas 0.5%/25% agrigel showed a more viscous phase. These properties could also be seen in Figure 5.3.2 Ei-ii. Both gels were stiffer than their agarose controls at 1Hz (G’-p<0.05).

Laminin staining of 25% matrigel showed that laminin forms a lattice in matrigel with many crevices (Figure 5.3.4). As the agarose concentration was increased the distance between each laminin strand increased. 0.7%/25% matrigel had large dark patches in between laminin strands that were not observed in 0.5%/25% matrigel meaning that its ECM formed a tighter lattice in the agarose. Therefore, 0.5%/25% matrigel scaffold was chosen for subsequent growth of bronchotubule structures.

Bronchotubule triple culture outlined in the Methods and in Section 5.2.0 was repeated using seeding densities of NHBE (450,000 cells/ml), NHLF (675,000 cells/ml) and NHASM (675,000 cells/ml) in 0.5%/25% agrigel. This resulted in the formation of bifurcating tubular structures that were not mimicked by control cells grown in 0.5% agarose (Figure 5.3.5). Bifurcating tubule growth was unidirectional and tubule structure was maintained for 20 days. However, beating cilia were not observed in the lumen.

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A B 1000 1000

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G’= 0.5% Agarose 0.5/25% Agrigel G’ = 0.7% Agarose 0.7/25% Agrigel G” = 0.5% Agarose 0.5/25% Agrigel G”= 0.7% Agarose 0.7/25% Agrigel

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Figure 5.3.3-Comparison of viscoelastic characteristics of agarose and agrigel: (A) Storage and loss modulus of 0.5% agarose compared to 0.5%/25% agrigel. (B) Storage and loss modulus of 0.7% agarose compared to 0.7%/25% agrigel. (C) Storage modulus of agarose at different concentrations measured at 1Hz. (D) Loss modulus of agarose at different concentrations measured at 1Hz. All data is presented as mean SEM of n=3 independent experiments using ANOVA compared to agarose. *p<0.05, **p<0.01, ***p<0.001.

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Figure 5.3.4-Laminin staining of Agrigel: 25% Matrigel stained positive for laminin (Texas red). Laminin was also detected in Agrigel mixes and was distributed homogenously throughout the agrigel for all agarose/matrigel mixes. Images are representative of n=3 independent experiments.

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Figure 5.3.5- Scaffold optimisation: Triple culture of normal healthy human bronchial epithelial cells (NHBE), normal healthy human lung fibroblasts (NHLF) and normal healthy human airway smooth muscle (NHASM) cells in different scaffolds. Cell seeding density; NHBE = 450,000 cells/ml, NHLF = 675,000 cells/ml and NHASM = 675,000 cells/ml. 10x magnification, scale bar = 100 μm. Images are representative of n=3 using cells from the same patient.

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5.5 NHLF and NHASM YFP and mCherry Transduction To track the migration and assembly of bronchotubules 100,000 NHLF and NHASM cells were transfected with YFP and mCherry respectively using lentiviral vectors. 100% of NHLF and NHASM cells were transduced by 306,900 and 311,200 viral particles respectively (Figure 5.4.1). The MOI was calculated at 3 for each cell type (see Methods section 2.8 for calculation).

A (NHLF) B (NHASM)

Figure 5.4.1-Stromal Cell Lentiviral Transduction: 100, 000 normal healthy human lung fibroblasts (NHLF) and normal healthy human airway smooth muscle (NHASM) cells were transfected with 306, 900 and 311, 200 viral particles respectively in 1 ml media. YFP and mCherry MOI= 3. 5x magnification, Scale bar is 50 μm. Images are representative of images from n=3 independent experiments.

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5.6 Agrigel Triple Culture Cells from 3 healthy epithelial cell donors were cultured with NHLF and NHASM in 0.5%/25% agrigel as previously described in the methods. NHLF (green) and NHASM (red) cells surrounded tubular epithelial structures (grey) showing that there was stromal-epithelial stratification within the tubules. NHLF and NHASM cultured independently of NHBE did not result in tubules unlike triple cultures (Figure 5.5.1 and 5.5.2).

The structures formed by each NHBE donor was different and tubule growth and change was continuous. Cultures were maintained for 20 days, however, no clear evidence of epithelial cell differentiation as represented by cilia formation was observed unlike that seen in bronchospheres in Chapter 3.

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Figure 5.5.1-Bronchotubule Triple Culture in 0.5/25% Agrigel: Triple culture of lentivirally transduced of normal healthy human lung fibroblasts (NHLF, green) and normal healthy human airway smooth muscle (NHASM, red) cells at 675,000 cells/ml respectively with normal healthy human bronchial epithelial cells (NHBE) at 450,000 cells/ml. NHLF and NHASM do not form tubules in agrigel compared to triple culture which form tubules over 20 days cultures. Magnification 10x, Scale bar is 100μm. Images are representative of those from n=3 different donors. 191

Figure 5.5.2 Bronchotubule Triple Culture in 0.5/25% Agrigel Day 20: Bronchotubules arise from spheroids and form bifurcating branches over 20 days culture. Normal healthy human lung fibroblasts (NHLF, green) and normal healthy human airway smooth muscle (HASM, red) cells surround and support the growth of normal healthy human bronchial epithelial cells (grey). Epithelial donors 420, 495 and 619 respectively. Images are representative of n=3 experiments. Scalebar = 100 μM. 192

Discussion

The data in this Chapter shows that cells of different lineages have significant morphological effects on organoid structures. Chapter 3 demonstrated the effect of matrigel and differentiation on basal epithelial cells. When these same healthy cell donors are cultured in 25% matrigel with stromal cells, organoid morphology turns from a sphere to budding, branching tubules which mimic early lung development.

Furthermore, tubule growth was shown to be epithelial cell-driven as has been observed in mouse embryonic models (Kumar et al., 2014, Schnatwinkel and Niswander, 2013, Samakovlis et al., 1996). Without the presence of epithelial cells bronchotubules did not form and lumens were not observed compared to stromal controls that remained as single cells (Figure 5.5.1 and 5.5.2). Therefore, the model shows that tubule size and luminal generation are epithelial cell-driven with tubule formation being dependent on epithelial-stromal cell crosstalk. There is a critical number of stromal and epithelial cells that are required for branching tubule formation as tubules could not develop when the stromal cell population was less than 150,000 cells/ml. However, luminal development relied on the presence of a higher epithelial cell population.

Furthermore, ECM proteins derived from the Matrigel were necessary for the development of tubules since tubule formation was not observed with triple cell culture in 0.5% agarose alone. Triple culture only formed tubules when matrigel was added to the agarose to form agrigel. Therefore, epithelial, stromal and ECM proteins must interact to form tubular structures and thus the bronchotubule culture may be mimicking cellular-ECM interactions observed during airway development (Metzger et al., 2008, Kumar et al., 2014). A recent study that grew NHBE cells in COPD ECM found changes in gene expression signatures from healthy NHBE to a more CHBE phenotype whereby the expression of genes such as tgfb1 were up-regulated showing that ECM gives important cues for cellular development (Hedstrom et al., 2018).

Models such as lung-on-a-chip (LOC) have recently been developed to model COPD as discussed in the Introduction (Section 1.5.2.1) (Huh et al., 2010, Benam et al., 2016). LOC relies on separating the cell layers from one another and whilst strict control of independent parameters such as blood flow and exposure of epithelial cells

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to air are major advantages, models such as these will not be suitable for developmental studies as the model cannot be adapted to incorporate parameters such as ECM or the addition of other cell types such as ASM. Lung development also occurs in a 3D environment that LOC cannot model which gives co-culture models grown in hydrogels an advantage for studies of airway and lung development. Also the separation of cell layers using a semipermeable membrane may have profound, yet unknown, physiological responses as seen with recent ECM studies (Hedstrom et al., 2018).

The data in this chapter further demonstrated that matrix mechanics has a major impact on cellular morphology. Bronchotubules collapsed in a soft 25% matrigel after 4-6 days, but in the stiffer Agrigel (Figures 5.3.2 E, 5.3.3 and 5.3.4), the structure could be maintained for 20 days (Figures 5.3.5, 5.5.1, 5.5.2). However, cilia formation was not observed in the tubules. Lack of basal cell differentiation in the tubules may be due to the change in the microenvironmental niche. The addition of stromal cells and the change in gel strength may cause cell differentiation to delay cilia formation. In human foetal airways, cilia formation does not occur until 11-24 gestational weeks (Carson et al., 2002). Therefore, cell-cell-ECM interaction (the joint interaction between cells together with the ECM) may have a temporal effect on differentiation. Bronchotubule experimental timepoints were based on experiments conducted in Chapter 3 where bronchospheres took ~18 days to differentiate from NHBE cells. Timepoints may need to be extended to allow cellular differentiation to occur in bronchotubules (see General Discussion Chapter 6).

Research is lacking on the effect of the biophysical environment on cell-cell communication. Whilst specific mechanisms were not elucidated in this study, it can be seen from Figure 5.6.1. that by changing the mechanical niche of the culture, tubules could survive longer. Furthermore, various studies have shown that cells can change phenotype depending on the mechanical stimuli from their matrix (Dupont et al., 2011, Krieg et al., 2008, Adamo et al., 2009). One particular study by Picolo et. al. demonstrated that by changing scaffold tension (elastic moduli of 0.7-1kPa), they could alter the fate of stem cells pushing them towards apoptosis, adipose and stromal cell fates. Furthermore, soft ECM scaffolds resulted in a cell geometry that was rounder, smaller and less spread compared to stiffer ECM structures (Dupont et al., 194

2011). In this instance the resulting increase in cellular contractile forces caused cellular stretching leading to larger, more spread and squarer cell morphology (Dupont et al., 2011).

These processes were shown to be governed by YAP/Tafazzin (TAZ) where their concentration and localisation was stimulated by the ECM tension which, in turn, altered the tension within the cellular cytoskeleton and stress fibres (Dupont et al., 2011). These observations in these types of studies may explain the longevity of the bronchotubule structure and the morphological change from a ganglionic morphology in 25% matrigel, to a bifurcating structure in 0.5%/25% agrigel.

However, agrigel presented technical problems. Mixing matrigel and agarose was difficult as the matrigel gelation point is 10°C and that of agarose is 36°C. The addition of agarose also made it difficult to visualise structures because light from the microscope was scattered increasing the background. Further studies are required to determine a better bio-unreactive gel or stiffening agent with which to make a matrigel composite (See Further studies section at end of General Discussion Chapter 6).

Another reason for the observed lack of differentiation could be the media itself. In the bronchotubule study, the growth factors and the type of medium were not optimised to allow comparison with the bronchosphere culture. A recent publication by Snoeck et al. where human pluripotent stem cells (hPSC) were differentiated into early lung budding organoids used eight different types of media during the course of the culture each with their individually determined growth factors (Chen et al., 2017).

In the bronchotubule study, the idea was to try to balance cell populations and the local microenvironment to enable a cell-cell-ECM signalling cascade that would permit cells to regulate each other to trigger morphological changes rather than through switching of media containing different exogenously added growth factors (Chen et al., 2017). The cells themselves were considered as reactants in a (bio)chemical/physical reaction. However, in vitro cultures will always require feeding via a medium especially for cell survival post seeding and before cell aggregation. Whilst the mechanical environment was optimised the culture may require optimisation of the differentiation medium which was derived from the bronchosphere experiments in Chapter 3 to better support the tubule culture.

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The bronchotube model involved using three different cell types that came from different donors which could also influence differentiation. The internal cellular chemistry and the release of distinct factors/analytes from each donor could be different and may present a barrier to the reproducibility of the culture. Whilst epithelial cell donors in the culture were varied, epithelial cells from each donor resulted in the successful formation of tubules. However, the stromal cell donors were not varied as the emphasis of the study was on the behaviour of epithelial progenitor cells and due to time constraints. Therefore, it is possible that if the experiment were to be repeated with stromal cells from other donors, different results could be obtained. Donor-donor cell heterogeneity adds a further variable that can be eliminated by repeating the culture with cell types from the same donor (Gruber et al., 2006). Although technically challenging this remains the goal of this research in understanding tubule formation and in modelling lung development.

There is very little knowledge on the molecular developmental events in lung formation as it is morally objectionable to experiment on human embryos. Thus, there is a gap where more human in vitro models are required to model lung development. This study shows that organoids can be further developed to investigate cell-cell-ECM signalling to create models to further understanding into developmental biology of the lung (see General discussion Chapter 6).

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6 Chapter 6

General Discussion

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6.0 General Discussion The hypothesis posed at the start of this thesis that CHBE cells would form phenotypically perturbed bronchospheres and that CSC treatment of NHBE cells would push their bronchosphere morphology toward a CHBE-B phenotype compared to NHBE-B phenotype was confirmed in Chapter 3. CHBE-B displayed airway remodelling showing mucus hypersecretion, reduced ciliogenesis and a developmental delay that mimicked previous published in vivo data (Barnes et al., 2015, Gao et al., 2015). CSC treatment hampered the development of NHBE bronchospheres causing developmental delay and inhibiting luminal cell population differentiation.

The factors leading to these phenotypes were further explored by RNA-seq that suggested that various cells such as club cells found in the lower airways may be present in NHBE-B, but not in CHBE-B. Furthermore, CHBE-B showed down regulation in major cell differentiation gene foxa2 previously shown to inhibit differentiation of distal epithelial cells (Wan et al., 2005). Transcriptomic profiling of CSC-NHBE-B was also closer to that of CHBE-B than NHBE-B after 20 days of culture suggesting that CSC-NHBE-B progenitor population was switching from NHBE to CHBE phenotype and that this may affect ciliary epithelial cell differentiation. However, RNA-seq results did show an up-regulation in muc5b and bpif1a2 transcript expression suggesting the presence of secretory cell subpopulations. Therefore, it is possible that differentiation may have occurred, but not of the classical cell populations that were assayed. Future bioinformatic analysis of the RNA-seq data using gene signature for specific airway epithelial cell subtypes may reveal the true differentiation status of these cells.

In both CSC-NHBE-B and CHBE-B PI3K control of FGFR1-4 signalling pathway were down-regulated by day 20 of culture. The FGF protein pathway is crucial for lung development and repair particularly that of the epithelium (Tong et al., 2016, Quantius et al., 2016). The FGF pathway interacts with many other developmental pathways such as Wnt, Notch and BMP that are required for correct cell differentiation. These pathways have also been shown to be down-regulated or altered in smokers and in COPD (Wang et al., 2011, Mori et al., 2015, Zong et al., 2016) . Therefore, down-

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regulation of the FGF pathway may show that there is dysfunctional cell homeostasis resulting in incorrect regeneration of epithelial cell populations (Volckaert and De Langhe, 2015, Fox et al., 2015). Thus, further, characterisation assessing FGF, FOXA2, Notch and Wnt pathways should be done to determine the mechanism behind ectopic cell population generation.

RNA-seq analysis revealed up-regulation of oxidative stress pathways for all conditions and during all days of culture assayed. Therefore, it is likely this is a feature of this culture method itself. Interestingly, the uterine environment is hypoxic with the developing foetus exposed to low levels of oxygen tension (<3%) rather than the 21% used in culture conditions (van Tuyl et al., 2005). Recent studies have shown that oxygen tension may have an effect on the differentiation of progenitor cells (van Tuyl et al., 2005). Therefore, the effect of oxidative stress may be masked by the 21% oxygen environment used in culture. As organoids are a developmental model these results may indicate that the culture may require mimicking of different oxygen tensions seen during development to improve the efficacy of the model in faithfully regenerating cell populations of the airway.

This study was limited by the number of patient samples that could be obtained. Donor to donor variability can give skewed results and so studies using donor cells require high number of samples. Experiments should be repeated with another 6 patients to increase ‘n’ numbers to 10 to allow for power calculations to determine adequate number of samples for complete characterisation of the bronchosphere model. Interestingly, the RNA-seq data was able to detect highly significant differences with n=4 samples.

In the presence of stromal cells, epithelial cells formed tubular branching structures. Bronchotubules initially exhibit similar features to bronchospheres where cells aggregate in to spheres. The heterogenous cell spheres then begin to branch as observed in mouse foetal organogenesis. In 25% matrigel, adjacent tubules merge as seen with bronchospheres. In the stiffer 0.5/25% agrigel, tubules could not merge probably due to the physical separation via the matrix. Due to time constraints, this aspect of the model was not fully characterised. Furthermore, IF labelling and q PCR analysis of cell populations such as ciliated, goblet and basal cells are needed to show

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that epithelial cells remain epithelial and/or differentiate. Initial visualisation under a light microscope did not show any beating cilia, however it was difficult to visualise the model through the agrigel scaffold. Further experiments are also needed to asses ASM functionality using muscle agonists. Development of this feature may allow for pharmacological testing of drugs that target the smooth muscle in lung obstructive diseases such as asthma. Bioinformatic analysis of RNA-seq data from tubules may be able to address many of these questions.

Bronchotubule culture does not reach a state of quiescence that is observed in the lung. The continued branching may be the reason for the lack of differentiation observed in the lumens of the tubules. The lung in vivo ends in alveolar saccules, formed by SOX9/ID2+ protein cells that differentiate to form ATII cells that further differentiate to form ATI cells. This SOX9/ID2+ cell population are theorised to be in the branching tip of the outgrowing lung tubule (Rockich et al., 2013). The addition of ATII cells could give the stop signal necessary to stop the branching and initiate differentiation.

Bronchotubule culture optimisation showed the importance of the mechanical effects of the viscoelastic ECM microenvironmental niche on organogenesis and cellular development. Although agrigel allowed for the extension of the life of the bronchotubules allowing for branching and cellular aggregation, it was technically demanding to use and provided poor visibility. The data gleaned from viscoelasticity experiments will allow for the creation of a better less technically demanding and more visually accessible composite gel scaffold. Properties to consider when choosing a new gel will be level of ease of technical manipulation, biodegradability, mechanical strength and viscoelasticity. Any cell culture method that is developed should be simple to execute whilst being conducted quickly and efficiently to ensure cells are not out of culture for a prolonged period.

Multiple biomimetic gels already exist for cell culture that are currently being used including poly-ethylene glycol (PEG), poly-ethylene glycol diacrylate (PEGDA) and polyglycolide (PGA) (Hu et al., 2012, Gasperini et al., 2014). The advantage of these gels is that they are liquid at room temperature and would therefore be easy to mix with matrigel. They can also be gelled via photo-crosslinking giving greater control

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over the timepoint of gelation. The gel viscoelasticity can be reduced by longer exposure time that give greater control over the stiffness of the scaffold whereas with agrigel, that uses thermal crosslinking, multiple agarose-matrigel composites must be created where agarose stock must be maintained above 36°C and matrigel below 10°C during culture. Photocrosslinking involves exposing gels with photoinitiators such as riboflavin, camphorquinone or fluorescein sodium salt to visible light for very short periods of time e.g. <1minute (Hu et al., 2012, Gasperini et al., 2014). Visible light has the added advantage of passing through tissue without causing damage. For example, blue light is already in use in clinical practice such as dentistry to solidify fillings.

Alternatively, instead of using matrigel as the main ECM gel, ECM could be obtained from healthy and COPD decellularized lung scaffolds of donors. The advantage of using this ECM is that it would be human ECM rather than murine ECM and cellular tissue could be matched to the ECM donor.

Other culture conditions that need to be optimised are oxygen tension of the culture and the supporting medium. Although the idea of the culture is to create a model where different cell populations support each other, missing features such as vasculature and immune cells will undoubtedly mean that supporting growth factors may be needed. Media from novel protocols such as the lung budding organoid (LBO) culture could be tested to determine the optimal conditions for culture (Narita et al., 2017).

Post-bronchotubule model optimisation, bronchotubule culture can be repeated using COPD-derived primary cells. RNA-seq of CHBE-B at day 20 showed down regulation of development pathways necessary for branching morphogenesis such as muscle. This may indicate that CHBE-B epithelial cells may generate aberrantly branching tubules due to dysregulated cell-cell signalling. Bronchotubules containing CHBE: COPD HLF (CHLF): COPD ASM (CHASM) compared to NHBE: NHLF: NHASM bronchotubules and the characterisation of cellular lineages, secreted matrix layers and scaffold remodelling could be analysed by RNA-seq and protein arrays/SomaLogic aptamer arrays. These approaches may delineate novel developmental genes and key pathways important in cell-cell signalling in COPD.

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Furthermore, disease-healthy cell hybrid bronchotubule models could be created e.g. NHBE: CHLF: CHASM or CHBE: NHLF: NHASM to elucidate cell lineages that are the drivers of abnormal developmental processes driving disease and whether replacing the disease driving lineage with a healthy lineage can be reparative. These kinds of experiments can allow for direct identification of cell lineages to target and aid in the discovery of novel pharmaceutical agents.

The effect of changes in scaffold mechanics on the lung could also potentially be studied using this model. Diseases such as COPD and IPF symptomatically display matrix stiffening. Growing disease cells in softer gels and healthy tubules in stiffer gels could model the effect of matrix stiffness on cell-cell signalling and resultant tubule morphology.

However, to use this model to its full potential, primary cell types from the same patient would need to be acquired and banked which may pose a barrier for statistical power acquisition in the regular use of the technique. Whilst in this thesis cells from different patients were able to generate bronchotubules, donor-donor variability may cause the culture to fail during triple culture of bronchotubules with cells from 3 different donors.

The lung is a complicated organ with multiple interacting cell types that ensure its function and homeostasis. These features make the lung a hard organ to study and replicate in a laboratory-controlled environment. The aim of this thesis was to create a 3D model that would replicate COPD phenotype in vitro (summarised in Figure 6.1). After the development of bronchospheres that were able to model COPD in vitro, incorporate other physiological features to try to model the complex cellular interaction of the airway resulting in a model that was able to replicate branching morphogenesis. The further advancement of models like these are crucial to push the understanding in lung disease development and pathogenesis. Through these experiments, it is evident that progenitor cells from disease such as COPD become reprogrammed due to continued damage from external agents such as cigarette smoke and generate perturbed dysfunctional tissues. The success and limitations of the thesis are summarised below.

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Successes:

• Bronchosphere culture with healthy or COPD HBE replicates structure of healthy or COPD airway epithelium. • Cigarette smoke treatment of healthy cells may push sphere phenotype to COPD. • Stromal cells induce formation of branching tubular structures rather than spheroidal structures. • Stiffening of ECM allowed for longer bronchotubule survival.

Limitations:

• RNAseq results of bronchospheres remain to be validated. • Agrigel scaffold enabled survival of bronchotubules, but was not good for imaging. • Bronchotubule culture requires cells to originate from the same donor. • Bronchotubule culture required high seeding density.

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A) Healthy Bronchospheres Develop in 20 Days

Day 20

Day 14 Day 8 Day 0 Day 2

B) COPD Bronchosphere Development is Delayed, and Cell Population is Secretory

C) Cigarette Smoke treated Healthy Cells form Abnormal Spheroids and Do Not Differentiate

D) Stromal Cells Induce Tubular Formation of Healthy Epithelial Cells

Figure 6.1-Summary of models: Healthy bronchospheres develop in 20 days and have a differentiated lumen, A, COPD bronchospheres show delay in development and have a higher secretory cell population leading to excess mucous production, B, cigarette smoke treatment of healthy cells results in abnormal spheroid development with multiple lumens, C, stromal cells induce branching morphogenesis of healthy epithelial cells and form tubules that do not have an undifferentiated lumen, D.

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7 Chapter 7

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