• Development and Reproduction
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• v.17 no.1
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• pp.9-16
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• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.5
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• pp.357-365
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• 2022

Simultaneous myofibril and mitochondrial development is crucial for the cardiac differentiation of pluripotent stem cells (PSCs). Specifically, mitochondrial energy metabolism (MEM) development in cardiomyocytes is essential for the beating function. Although previous studies have reported that MEM is correlated with cardiac differentiation, the process and timing of MEM regulation for cardiac differentiation remain poorly understood. Here, we performed transcriptome analysis of cells at specific stages of cardiac differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected MEM genes strongly upregulated at cardiac lineage commitment and in a time-dependent manner during cardiac maturation and identified the protein-protein interaction networks. Notably, MEM proteins were found to interact closely with cardiac maturation-related proteins rather than with cardiac lineage commitment-related proteins. Furthermore, MEM proteins were found to primarily interact with cardiac muscle contractile proteins rather than with cardiac transcription factors. We identified several candidate MEM regulatory genes involved in cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpα, and Cdkn2a in mESC-derived cells, and CCK and NOS3 in hiPSC-derived cells) and cardiac maturation (Ppargc1α, Pgam2, Cox6a2, and Fabp3 in mESC-derived cells, and PGAM2 and SLC25A4 in hiPSC-derived cells). Therefore, our findings show the importance of MEM in cardiac maturation.

• Development and Reproduction
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• v.26 no.4
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• pp.155-163
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• 2022

Human pluripotent stem cells (hPSCs) can give rise to a vast array of differentiated derivatives, which have gained great attention in the field of in vitro toxicity evaluation. We have previously demonstrated that hPSC-derived alveolar epithelial cells (AECs) are phenotypically and functionally similar to primary AECs and could be more biologically relevant alternatives for assessing the potential toxic materials including in fine dust and cigarette smoking. Therefore, in this study, we employed hPSC-AECs to evaluate their responses to exposure of various concentrations of diesel particulate matter (dPM), cigarette smoke extract (CSE) and nicotine for 48 hrs in terms of cell death, inflammation, and oxidative stress. We found that all of these toxic materials significantly upregulated the transcription of pro-inflammatory cytokines such as IL-1α, IL-β, IL-6, and TNF-α. Furthermore, the exposure of dPM (100 ㎍/mL) strongly induced upregulation of genes related with cell death, inflammation, and oxidative stress compared with other concentrations of CSE and nicotine. These results suggest that hPSC-AECs could be a robust in vitro platform to evaluate pulmotoxicity of various air pollutants and harmful chemicals.

• BMB Reports
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• v.51 no.2
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• pp.85-91
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• 2018

Pluripotent stem cell (PSC) variations can cause significant differences in the efficiency of cardiac differentiation. This process is unpredictable, as there is not an adequate indicator at the undifferentiated stage of the PSCs. We compared global gene expression profiles of two PSCs showing significant differences in cardiac differentiation potential. We identified 12 up-regulated genes related to heart development, and we found that 4 genes interacted with multiple genes. Among these genes, Gata6 is the only gene that was significantly induced at the early stage of differentiation of PSCs to cardiomyocytes. Gata6 knock-down in PSCs decreased the efficiency of cardiomyocyte production. In addition, we analyzed 6 mESC lines and 3 iPSC lines and confirmed that a positive correlation exists between Gata6 levels and efficiency of differentiation into cardiomyocytes. In conclusion, Gata6 could be utilized as a biomarker to select the best PSC lines to produce PSC-derived cardiomyocytes for therapeutic purposes.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.209-214
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• 2010

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

• International Journal of Oral Biology
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• v.37 no.4
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• pp.175-180
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• 2012

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest-related precursor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leukemia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our present study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT-PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c-Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog expression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stemness in SKPs through the suppression of lineage differentiation and in part through the control of cell proliferation.

• Molecules and Cells
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• v.44 no.10
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• pp.746-757
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• 2021

Plant somatic cells can be reprogrammed into a pluripotent cell mass, called callus, which can be subsequently used for de novo shoot regeneration through a two-step in vitro tissue culture method. MET1-dependent CG methylation has been implicated in plant regeneration in Arabidopsis, because the met1-3 mutant exhibits increased shoot regeneration compared with the wild-type. To understand the role of MET1 in de novo shoot regeneration, we compared the genome-wide DNA methylomes and transcriptomes of wildtype and met1-3 callus and leaf. The CG methylation patterns were largely unchanged during leaf-to-callus transition, suggesting that the altered regeneration phenotype of met1-3 was caused by the constitutively hypomethylated genes, independent of the tissue type. In particular, MET1-dependent CG methylation was observed at the blue light receptor genes, CRYPTOCHROME 1 (CRY1) and CRY2, which reduced their expression. Coexpression network analysis revealed that the CRY1 gene was closely linked to cytokinin signaling genes. Consistently, functional enrichment analysis of differentially expressed genes in met1-3 showed that gene ontology terms related to light and hormone signaling were overrepresented. Overall, our findings indicate that MET1-dependent repression of light and cytokinin signaling influences plant regeneration capacity and shoot identity establishment.

• BMB Reports
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• v.51 no.5
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• pp.242-248
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• 2018

Induced pluripotent stem cells (iPSCs) show great promise for replacing current stem cell therapies in the field of regenerative medicine. However, the original method for cellular reprogramming, involving four exogenous transcription factors, is characterized by low efficiency. Here, we focused on using epigenetic modifications to enhance the reprogramming efficiency. We hypothesized that there would be a new reprogramming factor involved in DNA demethylation, acting on the promoters of pluripotency-related genes. We screened proteins that bind to the methylated promoter of Oct4 and identified Zinc finger protein 127 (Zfp127), the functions of which have not yet been identified. We found that Zfp127 binds to the Oct4 promoter. Overexpression of Zfp127 in fibroblasts induced demethylation of the Oct4 promoter, thus enhancing Oct4 promoter activity and gene expression. These results demonstrate that Zfp127 is a novel regulator of Oct4, and may become a potent target to improve cellular reprogramming.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.232-239
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• 2019

In general, cloned pigs have been produced using the somatic cell nuclear transfer (SCNT) technique with various types of somatic cells; however, the SCNT technique has disadvantages not only in its low efficiency but also in the development of abnormal clones. This study aimed to compare early embryonic development and quality of SCNT embryos with those of induced pluripotent stem cells (iPSCs) NT embryos (iPSC-NTs). Ear fibroblast cells were used as donor cells and iPSCs were generated from these cells by lentiviral transduction with human six factors (Oct4, Sox2, c-Myc, Nanog, Klf4 and Lin28). Blastocyst formation rate in iPSC-NT (23/258, 8.9%) was significantly lower than that in SCNT (46/175, 26.3%; p < 0.05). Total cell number in blastocysts was similar between two groups, but blastocysts in iPSC-NT had a lower number of apoptotic cells than in SCNT (2.0 ± 0.6 vs. 9.8 ± 2.9, p < 0.05). Quantitative PCR data showed that apoptosis-related genes (bax, caspase-3, and caspase-9) were highly expressed in SCNT than iPSC-NT (p < 0.05). Although an early development rate was low in iPSC-NT, the quality of cloned embryos from porcine iPSC was higher than that of embryos from somatic cells. Therefore, porcine iPSCs could be used as a preferable cell source to create a clone or transgenic animals by using the NT technique.

• Molecules and Cells
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• v.45 no.12
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• pp.923-934
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• 2022

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in applications such as regenerative medicine, cardiac disease modeling, and in vitro drug evaluation. However, hPSC-CMs are immature, which limits their applications. During development, the maturation of CMs is accompanied by a decline in their proliferative capacity. This phenomenon suggests that regulating the cell cycle may facilitate the maturation of hPSC-CMs. Aurora kinases are essential kinases that regulate the cell cycle, the role of which is not well studied in hPSC-CM maturation. Here, we demonstrate that CYC116, an inhibitor of Aurora kinases, significantly promotes the maturation of CMs derived from both human embryonic stem cells (H1 and H9) and iPSCs (induced PSCs) (UC013), resulting in increased expression of genes related to cardiomyocyte function, better organization of the sarcomere, increased sarcomere length, increased number of mitochondria, and enhanced physiological function of the cells. In addition, a number of other Aurora kinase inhibitors have also been found to promote the maturation of hPSC-CMs. Our data suggest that blocking aurora kinase activity and regulating cell cycle progression may promote the maturation of hPSC-CMs.