• Journal of Animal Reproduction and Biotechnology
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• v.38 no.4
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• pp.199-208
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• 2023

Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.

• 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.

• Molecules and Cells
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• v.37 no.1
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• pp.31-35
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• 2014

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.

• Journal of Genetic Medicine
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• v.9 no.2
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• pp.67-72
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• 2012

The generation of induced pluripotent stem cells (iPSCs) derived from patients' somatic cells provides a new paradigm for studying human genetic diseases. Human iPSCs which have similar properties of human embryonic stem cells (hESCs) provide a powerful platform to recapitulate the disease-specific cell types by using various differentiation techniques. This promising technology has being realized the possibility to explore pathophysiology of many human genetic diseases at the molecular and cellular levels. Furthermore, disease-specific human iPSCs can also be used for patient-based drug screening and new drug discovery at the stage of the pre-clinical test in vitro. In this review, we summarized the concept and history of cellular reprogramming or iPSC generation and highlight recent progresses for disease modeling using patient-specific iPSCs.

• Journal of Life Science
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• v.29 no.8
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• pp.895-903
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• 2019

The aim of the present study was to improve the cell viability of human dental papilla derived single-induced pluripotent stem cells (iPSCs) using a Rho-associated protein kinase (ROCK) inhibitor, Y-27632. The iPSCs were produced using an episomal plasmid-based reprogramming method. After cell separation using trypsin, the iPSCs were treated with 0, 0.5, 1, 2.5, 5, 7.5, or $10{\mu}M$ Y-27632 for 5 d. Cell viability increased significantly following the $5{\mu}M$ Y-27632 treatment (p<0.05). When the iPSCs were exposed to medium containing $10{\mu}M$ Y-27632 for 0, 1, 2, 3, 4, and 5 d, the cell viability rate increased significantly in accordance with the cell viability rate (p<0.05). To evaluate the effect of the Y-27632 treatment on stemness characteristics, the expression of stem cell-specific transcripts and telomerase activity were investigated in the iPSCs treated with $10{\mu}M$ Y-27632 for 5 d. The expression levels of stem cell-specific transcripts, such as OCT-4, NONOG, and SOX-2, and telomerase activity were not significantly different in the iPSCs treated with $10{\mu}M$ Y-27632 as compared with those of untreated control iPSCs (p>0.05). Taken together, the results demonstrated that cell viability can be improved by treatment with the ROCK inhibitor Y-27632, without losing iPSC stemness characteristics.

• BMB Reports
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• v.55 no.9
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• pp.453-458
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• 2022

Diabetes mellitus (DM) is a serious disease in which blood sugar levels rise abnormally because of failed insulin production or decreased insulin sensitivity. Although many studies are being conducted for the treatment or early diagnosis of DM, it is not fully understood how mitochondrial genome (mtDNA) abnormalities appear in patients with DM. Here, we induced iPSCs from fibroblasts, PBMCs, or pancreatic cells of three patients with type 2 DM (T2D) and three patients with non-diabetes counterpart. The mtDNA mutations were detected randomly without any tendency among tissues or patients. In T2D patients, 62% (21/34) of iPSC clones harbored multiple mtDNA mutations, of which 37% were homoplasmy at the 100% mutation level compared to only 8% in non-diabetes. We next selected iPSC clones that were a wild type or carried mutations and differentiated into pancreatic cells. Oxygen consumption rates were significantly lower in cells carrying mutant mtDNA. Additionally, the mutant cells exhibited decreased production of insulin and reduced secretion of insulin in response to glucose. Overall, the results suggest that screening mtDNA mutations in iPSCs from patients with T2D is an essential step before pancreatic cell differentiation for disease modeling or autologous cell therapy.

• 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.

• BMB Reports
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• v.48 no.2
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• pp.68-80
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• 2015

G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of self-renewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs.

• Journal of Life Science
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• v.30 no.4
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• pp.394-401
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• 2020

As embryonic stem cells become pluripotent, they may cause tumor development when injected into a host. Therefore, researchers are focusing heavily on the therapeutic potential of tissue-specific stem cells (adult stem cells) without resultant tumor formation. Adult stem cells can proliferate for a limited number of generations and are restricted to certain cell types (multipotent). Mature tissue cell types in mammals cannot be intrinsically dedifferentiated or transdifferentiated to adult stem cells. Hence, the technology of induced pluripotent stem cells (iPSCs) for reprogramming adult somatic cells was introduced in 2006, ushering in a new era in adult stem cell research. Although iPSCs have been widely used in the field, the approach has several limitations: instability of the reprogramming process, risk of incomplete reprogramming, and exposure to transgenes integrated into the cell genome. Two years before the introduction of the iPSC technique, the synthetic small molecule 2,6-disubstituted purine, called reversine, was introduced. Reversine can induce the dedifferentiation of committed cells into multipotent progenitor-type cells by reprogramming and converting adult cells to other cell types under appropriate stimuli. Thus, it can be used as a chemically induced multipotent cell agent to overcome the limitations of iPSCs. Also, as an alternative therapeutic approach for treating obesity, it can be used to generate beige cells by browning white adipocytes. While reversine has the potential to act as an anti-cancer agent, this review focuses on its role in differentiation, dedifferentiation, and transdifferentiation in somatic cells.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.3
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• pp.267-275
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• 2023

Cardiotoxicity, particularly drug-induced Torsades de Pointes (TdP), is a concern in drug safety assessment. The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (human iPSC-CMs) has become an attractive human-based platform for predicting cardiotoxicity. Moreover, electrophysiological assessment of multiple cardiac ion channel blocks is emerging as an important parameter to recapitulate proarrhythmic cardiotoxicity. Therefore, we aimed to establish a novel in vitro multiple cardiac ion channel screening-based method using human iPSC-CMs to predict the drug-induced arrhythmogenic risk. To explain the cellular mechanisms underlying the cardiotoxicity of three representative TdP high- (sotalol), intermediate- (chlorpromazine), and low-risk (mexiletine) drugs, and their effects on the cardiac action potential (AP) waveform and voltage-gated ion channels were explored using human iPSC-CMs. In a proof-of-principle experiment, we investigated the effects of cardioactive channel inhibitors on the electrophysiological profile of human iPSC-CMs before evaluating the cardiotoxicity of these drugs. In human iPSC-CMs, sotalol prolonged the AP duration and reduced the total amplitude (TA) via selective inhibition of I_Kr and I_Na currents, which are associated with an increased risk of ventricular tachycardia TdP. In contrast, chlorpromazine did not affect the TA; however, it slightly increased AP duration via balanced inhibition of I_Kr and I_Ca currents. Moreover, mexiletine did not affect the TA, yet slightly reduced the AP duration via dominant inhibition of I_Ca currents, which are associated with a decreased risk of ventricular tachycardia TdP. Based on these results, we suggest that human iPSC-CMs can be extended to other preclinical protocols and can supplement drug safety assessments.