iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Summary

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Abstract

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC's, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Introduction

The blood-air barrier in the large airways includes the trachea, bronchi, and bronchioles. It plays a crucial role in maintaining respiratory health and is made up of the airway epithelium, basement membrane, blood vessels and endothelial cells, and immune cells. The primary epithelial cells in the airway encompass basal cells, club cells, ciliated cells, and goblet cells. Basal cells, acting as the stem cells of the airway epithelium, are multipotent progenitors with high proliferative and self-renewal capabilities, giving rise to mature airway epithelial cells¹. Club cells are non-ciliated, secretory cells that contribute to the maintenance o....

Protocol

This study protocol was approved by the Institutional Review Board of UCSD's Human Research Protections Program (181180). This protocol uses small molecules and growth factors to direct the differentiation of pluripotent stem cells into airway cells, endothelial cells, and macrophages. These cells are then co-cultured onto cell culture inserts and polarized in an air-liquid interface. The details of the reagents, consumables, and equipment used are listed in the Table of Materials. The media and buff.......

Discussion

The development and implementation of a model of the blood-air barrier in the large airways for studying viral infections and other toxins require meticulous attention to detail to ensure the successful differentiation and function of the various cell types involved. This discussion will address key factors for successful differentiation, potential challenges, alternative applications, and implications for studying human diseases.

To ensure a successful differentiation, attention to the type .......

Materials

Name	Company	Catalog Number	Comments
12 well plates	Corning	3512
12-well inserts, 0.4 µm, translucent	VWR	10769-208
2-mercaptoethanol	Sigma-Aldrich	M3148
Accutase	Innovative Cell Tech	AT104
Activin A	R&D Systems	338-AC
All-trans retinoic acid (RA)	Sigma-Aldrich	R2625
ascorbic acid	Sigma	A4544
B27 without retinoic acid	ThermoFisher	12587010
BMP4	R&D Systems	314-BP/CF
Bovine serum albumin (BSA) Fraction V, 7.5% solution	Gibco	15260-037
Br-cAMP	Sigma-Aldrich	B5386
CD 14 (FITC)	BioLegend	982502
CD 31 PECAM-1(APC)	R&D System	FAB3567A
CD 45 (PE)	BD Biosciences	560975
CD 68 (PE)	BioLegend	33808
CHIR99021	Abcam	ab120890
CPM	Fujifilm	014-27501
Dexamethasone	Sigma-Aldrich	D4902
Dispase	StemCellTech	7913
DMEM/F12	Gibco	10565042
Dorsomorphin	R&D Systems	3093
E-CAD/CD 324 (APC)	BioLegend	324107
EGF	R&D Systems	236-EG
EGM2 Medium	Lonza	CC-3162
EPCAM/CD 326 (APC)	BioLegend	324212
FBS	Gibco	10082139
FGF10	R&D Systems	345-FG/CF
FGF7	R&D Systems	251-KG/CF
Fibronectin	Fisher	356008
Forskolin	Abcam	ab120058
Glutamax	Life Technologies	35050061
Ham’s F12	Invitrogen	11765-054
HEPES	Gibco	15630-080
IBMX (3-Isobtyl-1-methylxanthine)	Sigma-Aldrich	I5879
IL-3	Peprotech	200-03
Iscove’s Modified Dulbecco’s Medium (IMDM) + Glutamax	Invitrogen	31980030
Knockout Serum Replacement (KSR)	Life Technologies	10828028
Matrigel	Corning	354230
M-CSF	Peprotech	300-25
Monothioglycerol	Sigma	M6145
mTeSR plus Kit (10/case)	Stem Cell Tech	5825
N2	ThermoFisher	17502048
NEAA	Life Technologies	11140050
PBS	Gibco	10010023
Pen/strep	Lonza	17-602F
ReleSR	Stem Cell Tech	5872
RPMI1640 + Glutamax	Life Technologies	12633012
SB431542	R&D Systems	1614
SCF	PeproTech	300-07
SMA	Invitrogen	50-9760-80
STEMdiff APEL 2 Medium	STEMCELL Technologies	5275
TrypLE Express	Gibco	12605-028
VEGF165	Preprotech	100-20
Vimentin	Cell Signaling	5741S
Y-27632 (Rock Inhibitor)	R&D Systems	1254/1
ZO-1	Invitrogen	339100