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Characterization of common marmoset dysgerminoma-like tumor induced by the lentiviral expression of reprogramming factors.

Yamaguchi S, Marumoto T, Nii T, Kawano H, Liao J, Nagai Y, Okada M, Takahashi A, Inoue H, Sasaki E, Fujii H, Okano S, Ebise H, Sato T, Suyama M, Okano H, Miura Y, Tani K - Cancer Sci. (2014)

Bottom Line: Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine.Prior to the clinical application of iPSCs, testing of their safety and usefulness must be carried out using reliable animal models of various diseases.In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c-MYC into CM fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Clinical Genetics, Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

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Dependence of common marmoset dysgerminoma-like (CM DG) cell growth on c-MYC and basic fibroblast growth factor (bFGF) signaling. (a) Inhibition of CM DG growth by knockdown of c-MYC. Cells (3 × 104) were seeded on 24-well plates and transduced with shRNA targeting OCT3/4, SOX2, KLF4, c-MYC, or all reprogramming factors (shAll). Cell growth curves were analyzed by cell counts at the indicated time points. Results are shown as means ± SD. ***P < 0.001. Nc, negative control (mock vector). (b) Growth rate of CM DGs was promoted by the addition of bFGF. Cells were cultured in the presence or absence (Nc) of bFGF. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. ***P < 0.001. (c) FGFR inhibitor suppressed CM DG growth. Cells were cultured in the presence or absence (Nc) of the FGFR1-4 inhibitor BGJ398; bFGF was added at 5 ng/mL. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. *P < 0.05. (d) CM DGs, aorta-gonado-mesonephros fibroblasts (AGM), and CM skin fibroblasts (SKIN) were treated with different concentrations of BGJ398 for 3 days, and the growth-inhibitory effects were analyzed by MTS assay. The IC50 for CM DGs was lower than those for parental AGM fibroblasts and control CM skin fibroblasts. Results are shown as means ± SD.
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fig05: Dependence of common marmoset dysgerminoma-like (CM DG) cell growth on c-MYC and basic fibroblast growth factor (bFGF) signaling. (a) Inhibition of CM DG growth by knockdown of c-MYC. Cells (3 × 104) were seeded on 24-well plates and transduced with shRNA targeting OCT3/4, SOX2, KLF4, c-MYC, or all reprogramming factors (shAll). Cell growth curves were analyzed by cell counts at the indicated time points. Results are shown as means ± SD. ***P < 0.001. Nc, negative control (mock vector). (b) Growth rate of CM DGs was promoted by the addition of bFGF. Cells were cultured in the presence or absence (Nc) of bFGF. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. ***P < 0.001. (c) FGFR inhibitor suppressed CM DG growth. Cells were cultured in the presence or absence (Nc) of the FGFR1-4 inhibitor BGJ398; bFGF was added at 5 ng/mL. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. *P < 0.05. (d) CM DGs, aorta-gonado-mesonephros fibroblasts (AGM), and CM skin fibroblasts (SKIN) were treated with different concentrations of BGJ398 for 3 days, and the growth-inhibitory effects were analyzed by MTS assay. The IC50 for CM DGs was lower than those for parental AGM fibroblasts and control CM skin fibroblasts. Results are shown as means ± SD.

Mentions: Common marmoset ARCs, CM ESCs, and iPS A cells derived from fetal liver cells (provided by Erika Sasaki, KEIO-REKEN Research Center for Human Cognition, Keio University, Tokyo, Japan) were maintained in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) containing 20% Knockout Serum Replacement (Gibco, Carlsbad, CA, USA), 0.1 mM non-essential amino acid (Gibco), 1 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1% antibiotic–antimycotics (Nacalai Tesque), 0.4 mM 2-mercaptoethanol (Sigma Aldrich), and 0.12% sodium hydroxide (Nacalai Tesque). The CM DGs were maintained in DMEM/F12 containing 10% FBS at 37°C in a 5% humidified CO2 atmosphere. Detailed descriptions of the cell culture, reprogramming method, and proliferation assay are provided in Figures 1 and 5.


Characterization of common marmoset dysgerminoma-like tumor induced by the lentiviral expression of reprogramming factors.

Yamaguchi S, Marumoto T, Nii T, Kawano H, Liao J, Nagai Y, Okada M, Takahashi A, Inoue H, Sasaki E, Fujii H, Okano S, Ebise H, Sato T, Suyama M, Okano H, Miura Y, Tani K - Cancer Sci. (2014)

Dependence of common marmoset dysgerminoma-like (CM DG) cell growth on c-MYC and basic fibroblast growth factor (bFGF) signaling. (a) Inhibition of CM DG growth by knockdown of c-MYC. Cells (3 × 104) were seeded on 24-well plates and transduced with shRNA targeting OCT3/4, SOX2, KLF4, c-MYC, or all reprogramming factors (shAll). Cell growth curves were analyzed by cell counts at the indicated time points. Results are shown as means ± SD. ***P < 0.001. Nc, negative control (mock vector). (b) Growth rate of CM DGs was promoted by the addition of bFGF. Cells were cultured in the presence or absence (Nc) of bFGF. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. ***P < 0.001. (c) FGFR inhibitor suppressed CM DG growth. Cells were cultured in the presence or absence (Nc) of the FGFR1-4 inhibitor BGJ398; bFGF was added at 5 ng/mL. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. *P < 0.05. (d) CM DGs, aorta-gonado-mesonephros fibroblasts (AGM), and CM skin fibroblasts (SKIN) were treated with different concentrations of BGJ398 for 3 days, and the growth-inhibitory effects were analyzed by MTS assay. The IC50 for CM DGs was lower than those for parental AGM fibroblasts and control CM skin fibroblasts. Results are shown as means ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317795&req=5

fig05: Dependence of common marmoset dysgerminoma-like (CM DG) cell growth on c-MYC and basic fibroblast growth factor (bFGF) signaling. (a) Inhibition of CM DG growth by knockdown of c-MYC. Cells (3 × 104) were seeded on 24-well plates and transduced with shRNA targeting OCT3/4, SOX2, KLF4, c-MYC, or all reprogramming factors (shAll). Cell growth curves were analyzed by cell counts at the indicated time points. Results are shown as means ± SD. ***P < 0.001. Nc, negative control (mock vector). (b) Growth rate of CM DGs was promoted by the addition of bFGF. Cells were cultured in the presence or absence (Nc) of bFGF. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. ***P < 0.001. (c) FGFR inhibitor suppressed CM DG growth. Cells were cultured in the presence or absence (Nc) of the FGFR1-4 inhibitor BGJ398; bFGF was added at 5 ng/mL. Cell numbers were counted at the indicated time points. Results are shown as means ± SD. *P < 0.05. (d) CM DGs, aorta-gonado-mesonephros fibroblasts (AGM), and CM skin fibroblasts (SKIN) were treated with different concentrations of BGJ398 for 3 days, and the growth-inhibitory effects were analyzed by MTS assay. The IC50 for CM DGs was lower than those for parental AGM fibroblasts and control CM skin fibroblasts. Results are shown as means ± SD.
Mentions: Common marmoset ARCs, CM ESCs, and iPS A cells derived from fetal liver cells (provided by Erika Sasaki, KEIO-REKEN Research Center for Human Cognition, Keio University, Tokyo, Japan) were maintained in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) containing 20% Knockout Serum Replacement (Gibco, Carlsbad, CA, USA), 0.1 mM non-essential amino acid (Gibco), 1 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1% antibiotic–antimycotics (Nacalai Tesque), 0.4 mM 2-mercaptoethanol (Sigma Aldrich), and 0.12% sodium hydroxide (Nacalai Tesque). The CM DGs were maintained in DMEM/F12 containing 10% FBS at 37°C in a 5% humidified CO2 atmosphere. Detailed descriptions of the cell culture, reprogramming method, and proliferation assay are provided in Figures 1 and 5.

Bottom Line: Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine.Prior to the clinical application of iPSCs, testing of their safety and usefulness must be carried out using reliable animal models of various diseases.In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c-MYC into CM fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Clinical Genetics, Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

Show MeSH
Related in: MedlinePlus