• 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.

• Molecules and Cells
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• v.46 no.4
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• pp.219-230
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• 2023

Down syndrome (DS) is the most common autosomal aneuploidy caused by trisomy of chromosome 21. Previous studies demonstrated that DS affected mitochondrial functions, which may be associated with the abnormal development of the nervous system in patients with DS. Runt-related transcription factor 1 (RUNX1) is an encoding gene located on chromosome 21. It has been reported that RUNX1 may affect cell apoptosis via the mitochondrial pathway. The present study investigated whether RUNX1 plays a critical role in mitochondrial dysfunction in DS and explored the mechanism by which RUNX1 affects mitochondrial functions. Expression of RUNX1 was detected in induced pluripotent stem cells of patients with DS (DS-iPSCs) and normal iPSCs (N-iPSCs), and the mitochondrial functions were investigated in the current study. Subsequently, RUNX1 was overexpressed in N-iPSCs and inhibited in DS-iPSCs. The mitochondrial functions were investigated thoroughly, including reactive oxygen species levels, mitochondrial membrane potential, ATP content, and lysosomal activity. Finally, RNA-sequencing was used to explore the global expression pattern. It was observed that the expression levels of RUNX1 in DS-iPSCs were significantly higher than those in normal controls. Impaired mitochondrial functions were observed in DS-iPSCs. Of note, overexpression of RUNX1 in N-iPSCs resulted in mitochondrial dysfunction, while inhibition of RUNX1 expression could improve the mitochondrial function in DS-iPSCs. Global gene expression analysis indicated that overexpression of RUNX1 may promote the induction of apoptosis in DS-iPSCs by activating the PI3K/Akt signaling pathway. The present findings indicate that abnormal expression of RUNX1 may play a critical role in mitochondrial dysfunction in DS-iPSCs.

• International Journal of Oral Biology
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• v.39 no.4
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• pp.177-185
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• 2014

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the $Neon^{TM}$ and $NEPA21^{TM}$ electroporators were tested. $Neon^{TM}$ electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and $NEPA21^{TM}$ electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by $Neon^{TM}$ electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

• KSBB Journal
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• v.25 no.3
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• pp.215-222
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• 2010

Monogenic diseases are resulted from modifications in a single gene of human cells. Because their treatment with pharmacological medicine have a temporary effect, continuous nursing care and retreatment are required. Gene therapy, gene targeting and induced pluripotent stem cell (iPSC) are considered permanent treatment methods of them. In gene therapy, however, retroviral vectors that have potential toxicity caused by random insertion of harmful virus are used as vehicles for transferring genetic materials. On the other hand, gene targeting could replace and remove the modified gene though homologous recombination (HR) induced by site-specific endonucleases. This short review provides a brief overview on the recently tailored endonucleses with high selectivity for HR.

• 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.

• Development and Reproduction
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• v.15 no.1
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• pp.41-52
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• 2011

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/$m{\ell}$ BMP4 or 0.01 ${\mu}M$ RA. In the BMP4-treated group, GFR-${\alpha}1$ and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR-${\alpha}1$ and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1 and immunocytochemistry using GFR-${\alpha}1$ antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.77-77
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• 1995

Human immunodeficiency virus type 1 (HIV-1) contains several nonstructural genes which are required for the viral replication and disease pathogenesis. Among them, tat and nef genes encode an essential transactivator of HIV-1 LTR and a pluripotent protein which seems to be essential for the in vivo but not in vitro viral replication, respectively. We constructed two tat and n of defective HIV-1 and tested for their ability to replicate in several T cells. The defective viruses did not replicate in CD4$\^$+/ T cells, but rescued in the recombinant Jurkat-tat cell which also contains tat gene. The replication of tat and nef defective HIV-1 which expresses chloramphenicol acetyltransferase(CAT) gene was easily detected by a sensitive CAT assay. No revertant was identified during the passages of the mutant viruses for more than two months in Jurkat-tat cells. tat and n of defective HIV-1 could be used instead of wild type viruse for several purposes such as inhibitor screening and development of attenuated AIDS vaccine.

• 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.

• Radiation Oncology Journal
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• v.9 no.1
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• pp.117-122
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• 1991

Malignant small round cell tumor (SRCT) of the thoracopulmonary region appears to originate in the soft tissues of the chest wall or the peripheral lung. A differential diagnosis of poorly differentiated small round cell tumors which include Ewing's sarcoma of bone and soft tissue, embryonal rhabdomyosarcoma, Askin tumor, neuroblastoma, peripheral neuroectodermal tumor, small cell osteogenic sarcoma and Iymphoma are often difficult by light microscopy alone. In recent, by the extensive studies electron microscopic examination, histochemical study, immune-chemical study, cytogenetics and gene analysis, these tumors may be derived from the primitive and pluripotential cells, differentiating into mesenchymal, epithelial and neural features in variable proportions. Treatment for SRCT of thoracopulmonary region is not determined because of massive involvement of the lung, pleura or soft tissues of the chest wall resulted in a dismal outcome despite aggressive surgery, irradiation and chemotherapy.

• BMB Reports
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• v.44 no.12
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• pp.793-798
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• 2011

Recently, pluripotency induction or cellular reprogramming by introducing critical transcription factors has been extensively studied, but has been demonstrated only in vitro. Based on reports that Oct4 is critically involved in transforming neural stem cells into pluripotent cells, we used the lentiviral vector to introduce the Oct4 gene into the hippocampal dentate gyrus (DG) of adult mice. We examined whether this manipulation led to cellular or behavioral changes, possibly through processes involving the transformation of NS cells into pluripotent cells. The Oct4 lentivirus-infused group and the green fluorescent protein lentivirus-infused group showed a similar thickness of the DG and a comparable level of synaptophysin expression in the DG. Furthermore, our behavioral analyses did not show any differences between the groups concerning exploratory activity, anxiety, or memory abilities. This first trial for pluripotency induction in vivo, despite negative results, provides implications and information for future studies on in vivo cellular reprogramming.