A Two-Dimensional Human Minilung System (Model) for Respiratory Syncytial Virus Infections
Abstract
:- (i)
- Standard methods of expansion and maintenance of hESCs
- (ii)
- Use of NOG (NOGGIN) instead of dorsomorphin
- (iii)
- Cell density in replatings
- (iv)
- Sizes of multiwell plates used
- (v)
- Final assembly of the minilungs on glass chamber slides
- (vi)
- Addition of primary lung fibroblasts to the generated epithelial cells
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
ACTA2 | Alpha Smooth Muscle Actin (α-SMA) |
AFE | Anterior Foregut Endoderm |
AQP5 | Aquaporin 5 |
ATI | Alveolar Type I Cells |
ATII | Alveolar Type II Cells |
BMP | Bone Morphogenic Protein |
CXCL10 | C-X-C Motif Chemokine Ligand 10 |
CXCL8 | C-X-C Motif Chemokine Ligand 8 (IL8) |
CX3CR1 | C-X3-C Motif Chemokine Receptor 1 |
DE | Definitive Endoderm |
dpi | Days Post-Infection |
DXD58 | DExd/H-Box Helicase 58 (RIG-1: Retinoic Acid Inducible Gene 1) |
EBs | Embryoid Bodies |
FGF | Fibroblast Growth Factor |
FGFR | Fibroblast Growth Factor Receptor |
FOXA2 | Forkhead Box A2 |
FOXJ1 | Forkhead Box J1 |
hESC | Human Embryonic Stem Cells |
HRSV | Human Respiratory Syncytial Virus |
IL1B IL6 | Interleukin 1 β Interleukin 6 |
ISG15 | ISG15 Ubiquitin-Like Modifier |
mAb | Monoclonal Antibody |
MEFs | Mouse Embryonic Fibroblasts |
moi | Multiplicity of Infection |
MUCIN5AC | Mucin 5AC, Oligomeric Mucus/Gel-Forming |
NKX2-1 | NK2 Homeobox 1 |
NOG | Noggin |
PDPN | Podoplanin |
pfu | Plaque Forming Unit |
PSC | Pluripotent Stem Cells |
qRT-PCR | Quantitative Real-Time RT-PCR (Reverse Transcription Polymerase Chain Reaction) |
SCGB1A1 | Secretoglobin Family 1A Member 1; CC10 |
SFTPA | Surfactant Protein A |
SFTPB | Surfactant Protein B |
SFTPC | Surfactant Protein C |
SFTPD | Surfactant Protein D |
SOX2 | SRY (Sex Determining Region Y)-box 2 |
TBP | TATA Box Binding Protein |
TGFB1 | Tumor Growth Factor β |
TNF | Tumor Necrosis Factor (TNF α) |
TP63 | Tumor Protein p63 |
WNT | Wingless-Related Integration Site |
μm | Micrometers |
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Day | Process | Atmosphere/Observations | Factors, Inhibitors and Hormones Used | Targeted Patwways Activation Inhibition |
---|---|---|---|---|
Matrigel depletion of MEFs | 95% air/5% CO2 | |||
0 | Primitive streak induction | 5% O2/95% N2/5% CO2 Low attachment plates | Wnt3a, BMP4, ROCK inhibitor | WNTs BMP apoptosis |
1–3 | Definitive endoderm induction | 5% O2/95% N2/5% CO2 Low attachment plates | Activin A, BMP4, hbFGF, ROCK inhibitor | ACVR BMP FGFR apoptosis |
4–6 | Anterior foregut induction | 5% O2/95% N2/5% CO2 fibronectin coated-plants | Dorsomorphin or NOGGIN, SB431542, IWP2 | BMP TGF-β WNTs |
6–15 | Lung progenitor induction and expansion | 5% O2/95% N2/5% CO2 (intermediate incubation under 95% air/5% CO2) fibronectin coated-plates | CHIR99021, KGF, FGF10, BMP4, EGF, all-trans retinoic acid | Wnts BMP FGFR2b (Ventralization) |
16–25 | Lung progenitor induction and expansion | 5% O2/95% N2/5% CO2 fibronectin coated-plates | CHIR99021, KGF, FGF10 | Wnts FGFR2b |
26–~1 year | Lung and airway epithelial maturation | 5% O2/95% N2/5% CO2 fibronectin coated-plates | CHIR99021, KGF, FGF10, Dexamethasone, IBMX, cAMP | Wnts FGFR2b (Alveolar maturation) |
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Magro-Lopez, E.; Guijarro, T.; Martinez, I.; Martin-Vicente, M.; Liste, I.; Zambrano, A. A Two-Dimensional Human Minilung System (Model) for Respiratory Syncytial Virus Infections. Viruses 2017, 9, 379. https://doi.org/10.3390/v9120379
Magro-Lopez E, Guijarro T, Martinez I, Martin-Vicente M, Liste I, Zambrano A. A Two-Dimensional Human Minilung System (Model) for Respiratory Syncytial Virus Infections. Viruses. 2017; 9(12):379. https://doi.org/10.3390/v9120379
Chicago/Turabian StyleMagro-Lopez, Esmeralda, Trinidad Guijarro, Isidoro Martinez, Maria Martin-Vicente, Isabel Liste, and Alberto Zambrano. 2017. "A Two-Dimensional Human Minilung System (Model) for Respiratory Syncytial Virus Infections" Viruses 9, no. 12: 379. https://doi.org/10.3390/v9120379