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Developmental Biology

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

Published: April 12th, 2021



1Department of Pediatrics, University of California, San Diego School of Medicine, 2Sanford Consortium for Regenerative Medicine, 3Sanford Burnham Prebys Medical Discovery Institute

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.

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.

The lung is a complicated, heterogeneous, dynamic organ that develops in six distinct stages - embryonic, pseudoglandular, canalicular, saccular, alveolar, and microvascular maturation1,2. 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. ....

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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........

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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.......

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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.......

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This research was supported by the California Institute for Regenerative Medicine (CIRM)  (DISC2-COVID19-12022).


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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

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