Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Summary

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Abstract

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFRα). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

Introduction

The lung is a complicated, heterogeneous, dynamic organ that develops in six distinct stages - embryonic, pseudoglandular, canalicular, saccular, alveolar, and microvascular maturation^1,2. The latter two phases occur pre and postnatally in human development while the first four stages occur exclusively during fetal development unless preterm birth occurs³. The embryonic phase begins in the endodermal germ layer and concludes with the budding of the trachea and lung buds. Lung development occurs in part via signaling between the epithelial and mesenchymal cells⁴. ....

Protocol

This study protocol was approved by the Institutional Review Board of UCSD's Human Research Protections Program (181180).

1. Definitive endoderm induction from induced pluripotent stem cells (Day 1 - 3)

1. Slowly thaw growth factor reduced (GFR)-basement membrane (BM) matrix medium on ice 30 minutes prior to use. In cold DMEM/F12, mixture, dilute the GFR BM matrix medium 1:1 such that it constitutes 50% of this medium. Place P1000 pipette tips in the freezer to chill prior to use.
2. Coat each well of a 12-well plate with 500 µL of 50% GFR-basement membrane matrix medium prepared in ice-cold DMEM/F12. Once the desir....

Results

24 hours after plating, day 1, iPSCs should be 50%-90% confluent. On day 2, DE should be 90%-95% confluent. During DE induction, it is common to observe significant cell death on day 4 but attached cells will retain a compact cobblestone morphology (Figure 2b). Discontinue differentiation if the majority of adherent cells detach and consider shortening exposure to DE media with activin A by 6-12 h. During AFE induction, cell death is minimal, and cells remain adherent, but will appear smalle.......

Discussion

The successful differentiation of 3D whole lung organoids (WLO) relies on a multi-step, 6-week protocol with attention to detail, including time of exposure to growth factors and small molecules, cellular density after passaging, and the quality of hiPSCs. For troubleshooting, see Table 2. hiPSCs should be approximately 70%-80% confluent, with less than 5% spontaneous differentiation prior to dissociation. This protocol calls for "mTeSR plus" medium; however,  plain "mTeSR" medium
.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell Culture
12 well plates	Corning	3512
12-well inserts, 0.4um, translucent	VWR	10769-208
2-mercaptoethanol	Sigma-Aldrich	M3148
Accutase	Innovative Cell Tech	AT104
ascorbic acid	Sigma	A4544
B27 without retinoic acid	ThermoFisher	12587010
Bovine serum albumin (BSA) Fraction V, 7.5% solution	Gibco	15260-037
Dispase	StemCellTech	7913
DMEM/F12	Gibco	10565042
FBS	Gibco	10082139
Glutamax	Life Technologies	35050061
Ham’s F12	Invitrogen	11765-054
HEPES	Gibco	15630-080
Iscove’s Modified Dulbecco’s Medium (IMDM) + Glutamax	Invitrogen	31980030
Knockout Serum Replacement (KSR)	Life Technologies	10828028
Matrigel	Corning	354230
Monothioglycerol	Sigma	M6145
mTeSR plus Kit (10/case)	Stem Cell Tech	5825
N2	ThermoFisher	17502048
NEAA	Life Technologies	11140050
Pen/strep	Lonza	17-602F
ReleSR	Stem Cell Tech	5872
RPMI1640 + Glutamax	Life Technologies	12633012
TrypLE	Gibco	12605-028
Y-27632 (Rock Inhibitor)	R&D Systems	1254/1
Growth Factors/Small Molecules
Activin A	R&D Systems	338-AC
All-trans retinoic acid (RA)	Sigma-Aldrich	R2625
BMP4	R&D Systems	314-BP/CF
Br-cAMP	Sigma-Aldrich	B5386
CHIR99021	Abcam	ab120890
Dexamethasone	Sigma-Aldrich	D4902
Dorsomorphin	R&D Systems	3093
EGF	R&D Systems	236-EG
FGF10	R&D Systems	345-FG/CF
FGF7	R&D Systems	251-KG/CF
IBMX (3-Isobtyl-1-methylxanthine)	Sigma-Aldrich	I5879
SB431542	R&D Systems	1614
VEGF/PIGF	R&D Systems	297-VP/CF
Primary antibodies			Dilution rate
CXCR4-PE	R&D Systems	FAB170P	1:200 (F)
HOPX	Santa Cruz Biotech	sc-398703	0.180555556
HTII-280	Terrace Biotech	TB-27AHT2-280	0.145833333
KRT5	Abcam	ab52635	0.180555556
NKX2-1	Abcam	ab76013	0.25
NKX2-1-APC	LS-BIO	LS-C264437	1:1000 (F)
proSPC	Abcam	ab40871	0.215277778
SCGB3A2	Abcam	ab181853	0.25
SOX2	Invitrogen	MA1-014	0.180555556
SOX9	R&D Systems	AF3075	0.180555556
SPB (mature)	7 Hills	48604	1: 1500 (F) 1:500 (W)a
SPC (mature)	LS Bio	LS-B9161	1:100 (F); 1:500 (W) a