• BMB Reports
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• v.45 no.12
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• pp.713-718
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• 2012

Gangliosides play important roles in the control of several biological processes, including proliferation and transmembrane signaling. In this study, we demonstrate the effect of ganglioside GM1 on the proliferation of mouse induced pluripotent stem cells (miPSCs). The proliferation rate of miPSCs was lower than in mouse embryonic stem cells (mESCs). Fluorescence activated cell sorting analysis showed that the percentage of cells in the G2/M phase in miPSCs was lower than that in mESCs. GM1 was expressed in mESCs, but not miPSCs. To confirm the role of GM1 in miPSC proliferation, miPSCs were treated with GM1. GM1-treated miPSCs exhibited increased cell proliferation and a larger number of cells in the G2/M phase. Furthermore, phosphorylation of mitogen-activated protein kinases was increased in GM1-treated miPSCs.

• BMB Reports
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• v.55 no.3
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• pp.142-147
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• 2022

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

• Development and Reproduction
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• v.21 no.1
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• pp.47-54
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• 2017

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, $100{\mu}M$), roscovitine (ROSC, $10{\mu}M$), or olomoucine (OLO, $200{\mu}M$) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 ($37.5{\pm}0.2%$), S ($34.0{\pm}0.6%$) and G2/M ($28.5{\pm}0.4%$). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.79-84
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• 2014

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibroblasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.