Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs
Abstract
:1. Introduction
2. Results
2.1. Differentiation and Characterization of hESCs into Epithelial-Like Stem Cells
2.2. Establishment of Differentiated Dental Epithelial-Like Stem Cell Lines Derived from hiPSC
2.3. Induction of Epithelial-Mesenchymal Transition (EMT) in Dental Epithelial-Like Stem Cell Lines Derived from hiPSCs
2.4. Differentiation Potential of Differentiated Dental Epithelial-Like Stem Cell Lines Derived From hiPSC
3. Discussion
4. Materials and Methods
4.1. Cell Preparation and Culture Conditions
4.2. Differentiation of Human ESCs and iPSCs into Epithelial-Like Cells
4.3. SV40 LT Transformation-Mediated Immortalization
4.4. Immunofluorescent Staining
4.5. Microsatellite (STR) Analysis
4.6. Total RNA Preparation and Reverse Transcription
4.7. Semi-Quantitative PCR
4.8. Flow Cytometry
4.9. Epithelial-Mesenchymal Transition (EMT) Induced by Transforming Growth Factor-β1 (TGF-β1)
4.10. Real-Time PCR
4.11. Odontogenic Differentiation
4.12. Western Blotting
4.13. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
Abbreviations
HERS/ERM | Hertwig’s epithelial root sheath/epithelial rests of Malassez |
hESCs | Human embryonic stem cells |
hiPSCs | Human induced pluripotent stem cells |
hdDPSC | Dental pulp stem cells from human exfoliated deciduous teeth |
EPI-ES EPI-iPSC | Dental epithelial-like stem cells derived from hESCs |
Dental epithelial-like stem cells derived from hiPSCs |
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HERS-SV40 | Human iPSCs | EPI-iPSC | |||
---|---|---|---|---|---|
STR Locus | Genotype | STR Locus | Genotype | STR Locus | Genotype |
D8S1179 | 13 | D8S1179 | 13 | D8S1179 | 13 |
D21S11 | 29, 30 | D21S11 | 28, 32.2 | D21S11 | 28, 32.2 |
D7S820 | 9, 11 | D7S820 | 7, 8 | D7S820 | 7, 8 |
CSF1PO | 11, 12 | CSF1PO | 12 | CSF1PO | 12 |
D3S1358 | 16, 17 | D3S1358 | 17 | D3S1358 | 17 |
TH01 | 7, 9 | TH01 | 9 | TH01 | 9 |
D13S317 | 9, 10 | D13S317 | 9, 12 | D13S317 | 9, 12 |
D16S539 | 12, 14 | D16S539 | 9, 12 | D16S539 | 9, 12 |
D2S1338 | 17,24 | D2S1338 | 18, 19 | D2S1338 | 18, 19 |
D19S433 | 14, 15.2 | D19S433 | 13, 14 | D19S433 | 13, 14 |
vWA | 14, 18 | vWA | 14, 16 | vWA | 14, 16 |
TPOX | 8, 9 | TPOX | 8, 11 | TPOX | 8, 11 |
D18S51 | 13, 15 | D18S51 | 13, 15 | D18S51 | 13, 15 |
AMEL | X, Y | AMEL | X, Y | AMEL | X, Y |
D5S818 | 10, 13 | D5S818 | 10, 11 | D5S818 | 10, 11 |
FGA | 26 | FGA | 22 | FGA | 22 |
Target Gene | 5′ Oligonucleotide | 3′ Oligonucleotide |
---|---|---|
GAPDH | GAT GCT GGC GCT GAG TAC G | GCT AAG CAG TTG GTG GTG C |
N-cadherin | ACA GTG GCC ACC TAC AAA GG | CCG AGA TGG GGT TGA TAA TG |
E-cadherin | TGC CCA GAA AAT GAA AAA GG | GTG TAT GTG GCA ATG CGT TC |
Vimentin | TCT ACG AGG AGG AGA TGC GG | GGT CAA GAC GTG CCA GAG AC |
ABCG2 | CCA CAG GTG GAG GCA AAT CT | TCG CGG TGC TCC ATT TAT CA |
EpCAM | GCT GGC CGT AAA CTG CTT TG | ACA TTT GGC AGC CAG CTT TG |
BMI1 | CAG CCC AGC AGG AGG TAT TC | GGA TGA GGA GAC TGC ACT GG |
P63 | ATG TTG TAC CTG GAA AAC AAT GC | GTG ATG GAG AGA GAG CAT CGA A |
P75 | ACC GAG CTG GAA GTC GAG | CTC ACC GCT GTG TGT GTA C |
SOX2 | GAC TTC ACA TGT CCC AGC AC | GGG TTT TCT CCA TGC TGT TT |
OCT4 | ACC CCT GGT GCC GTG AA | GGC TGA ATA CCT TCC CAA ATA |
NANOG | CCT ATG CCT GTG ATT TGT GG | TTC TCT GCA GAA GTG GGT TG |
Amelogenin | AAG AAC CAT CAA GAA ATG GGG | TGA TAT AAC CAG GGT GCC CA |
Enamelin | TCC ACG GAA ATC CTC AGC AC | GGG GGT TGA GCT TCC TCT TC |
KLK4 | GAT CGC TCG TCT CTG GTA GC | GAG TTC TGG AAA CAG TGT GCG |
Runx2 | CCC AGT ATG AGA GTA GGT GTC C | GGG TAA GAC TGG TCA TAG GAC C |
BSP | AAG GCT ACG ATG GCT ATG ATG GT | AAT GGT AGC CGG ATG CAA AG |
OCN | ATC CTT TGG GGT TTG GCC TAC | GCC AAT AGG GCG AGG AGT G |
DMP1 | ACA GGC AAA TGA AGA CCC | TTC ACT GGC TTG TAT GG |
MEPE | CAA GAA GCC AGG TAT TCT GAA GG | TGT GGT TGA AAT GTT GGT GCT |
Col1a1 | CAA AAA ATG GGA GAC AAT TTC ACA | TCA TGT TCG GTT GGT CAA AGA T |
DSPP | GCC AGA GCA AGT CTG GTA ACG GT | TGT CTC TGC AGG AGT TAG GTC TTG GT |
Components | Contents of α5 (Control Group) | Contents of α5OB (Differentiation Group) |
---|---|---|
Alpha MEM | - | - |
FBS | 5% | 5% |
Antibiotics-antimycotics | 1% | 1% |
Dexamethasone | - | 0.1 µM |
B-glycerophosphate | - | 10 mM |
L- ascorbic acid phosphate | - | 50 µg/mL |
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Kim, G.-H.; Yang, J.; Jeon, D.-H.; Kim, J.-H.; Chae, G.Y.; Jang, M.; Lee, G. Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs. Int. J. Mol. Sci. 2020, 21, 4384. https://doi.org/10.3390/ijms21124384
Kim G-H, Yang J, Jeon D-H, Kim J-H, Chae GY, Jang M, Lee G. Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs. International Journal of Molecular Sciences. 2020; 21(12):4384. https://doi.org/10.3390/ijms21124384
Chicago/Turabian StyleKim, Gee-Hye, Jihye Yang, Dae-Hyun Jeon, Ji-Hye Kim, Geun Young Chae, Mi Jang, and Gene Lee. 2020. "Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs" International Journal of Molecular Sciences 21, no. 12: 4384. https://doi.org/10.3390/ijms21124384
APA StyleKim, G.-H., Yang, J., Jeon, D.-H., Kim, J.-H., Chae, G. Y., Jang, M., & Lee, G. (2020). Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs. International Journal of Molecular Sciences, 21(12), 4384. https://doi.org/10.3390/ijms21124384