Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture and 3D Organoid Culture
2.2. IL-13 and UAB30 Treatments
2.3. Histology, Immunohistochemistry and Immunofluorescence
2.4. Immunoblotting
2.5. Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR)
2.6. Statistical Analyses
3. Results
3.1. Exogenous EGF Is Dispensable for Normal Human Esophageal Organoid Growth in ADF-Based Medium
3.2. Inhibition of TGFβ Receptor-Mediated Signaling or Rho-Associated Kinase (ROCK) Increases Organoid Formation Efficiency
3.3. HOME0 Permits Normal Esophageal Organoid Formation from Patient Biopsies
3.4. IL-13 Induces BCH-like Changes in Normal Esophageal Organoids Formed in HOME0
3.5. RXR Stimulation Induces BCH-like Changes in Normal Esophageal Organoids Formed in HOME0
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Abbreviation | Name |
---|---|
AI | Artificial Intelligence |
BCH | basal cell hyperplasia |
BMP | bone morphogenetic proteins |
DAPI | 4′,6-diamidino-2-phenylindole |
DMSO | Dimethyl sulfoxide |
EoE | eosinophilic esophagitis |
H&E | Hematoxylin and Eosin |
IF | immunofluorescence |
IHC | immunohistochemistry |
IL | interleukin |
IVL | Involucrin |
OFR | Organoid formation rate |
PBS | phosphate-buffered saline |
PDL | population doubling level |
PDO | Patient-derived organoids |
qRT-PCR | quantitative reverse-transcription polymerase chain reaction |
RAR | retinoic acid receptor |
RXR | retinoic-X receptor |
3D | three-dimensional |
TGF-β | transforming growth factor-beta |
Abbreviation | Name |
---|---|
ADF | advanced Dulbecco’s Modified Eagle Medium/Ham’s F-12 |
ADF3+ | advanced DMEM +/+/+ |
CM | conditioned medium |
CM-NR | CM containing Noggin and R-Spondin1 |
EGF | epidermal growth factor |
FGF2 | fibroblast growth factor 2 |
FGF10 | fibroblast growth factor 10 |
HB-EGF | heparin-binding EGF-like growth factor |
HOME | human organoid medium containing EGF at indicated concentrations |
HOME0 | human organoid medium containing 0 ng/mL EGF |
HOME0.1 | human organoid medium containing 0.1 ng/mL EGF |
HOME1 | human organoid medium containing 1 ng/mL EGF |
HOME10 | human organoid medium containing 10 ng/mL EGF |
HOME0 ΔA | HOME0 devoid of A83-01 |
KSFM | keratinocyte serum-free medium |
KSFMC | KSFM medium containing 0.6 mM Ca2+ |
MOM | murine organoid medium |
Name | ADF3+ | HOME | HOME | MOM | KSFMC |
---|---|---|---|---|---|
Base medium | ADF | ADF | ADF | ADF | KSFM |
B27 | (+) | (+) | (+) | (+) | U |
N2 | (−) | (+) | (+) | (+) | U |
NAC | (+) | (+) | (+) | (+) | U |
CM-NR | (+) | (+) | (+) | (+) | U |
EGF (ng/mL) | 50 † | 0 or 50 † | Various † (0–50) | 50 †† | 1 † |
A83-01 (µM) | 0.5 | 0 | 5 | 0 | U |
Nicotinamide | (+) | (−) | (−) | (−) | U |
FGF110 | (+) | (−) | (−) | (−) | U |
FGF2 | (+) | (−) | (−) | (−) | U |
CHI99021 | (+) | (−) | (−) | (−) | U |
Forskolin | (+) | (−) | (−) | (−) | U |
Prostaglandin E2 | (+) | (−) | (−) | (−) | U |
CaCl2 (mM) | 1 | 1 | 1 | 1 | 0.6 |
Y-27632 | (+) | (+) | (+) | (+) | (+) |
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Shimonosono, M.; Morimoto, M.; Hirose, W.; Tomita, Y.; Matsuura, N.; Flashner, S.; Ebadi, M.S.; Okayasu, E.H.; Lee, C.Y.; Britton, W.R.; et al. Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids. Biomolecules 2024, 14, 1126. https://doi.org/10.3390/biom14091126
Shimonosono M, Morimoto M, Hirose W, Tomita Y, Matsuura N, Flashner S, Ebadi MS, Okayasu EH, Lee CY, Britton WR, et al. Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids. Biomolecules. 2024; 14(9):1126. https://doi.org/10.3390/biom14091126
Chicago/Turabian StyleShimonosono, Masataka, Masaki Morimoto, Wataru Hirose, Yasuto Tomita, Norihiro Matsuura, Samuel Flashner, Mesra S. Ebadi, Emilea H. Okayasu, Christian Y. Lee, William R. Britton, and et al. 2024. "Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids" Biomolecules 14, no. 9: 1126. https://doi.org/10.3390/biom14091126