• International Journal of Stem Cells
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• v.17 no.2
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• pp.182-193
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• 2024

To address the limitations of animal testing, scientific research is increasingly focused on developing alternative testing methods. These alternative tests utilize cells or tissues derived from animals or humans for in vitro testing, as well as artificial tissues and organoids. In western countries, animal testing for cosmetics has been banned, leading to the adoption of artificial skin for toxicity evaluation, such as skin corrosion and irritation assessments. Standard guidelines for skin organoid technology becomes necessary to ensure consistent data and evaluation in replacing animal testing with in vitro methods. These guidelines encompass aspects such as cell sourcing, culture techniques, quality requirements and assessment, storage and preservation, and organoid-based assays.

• International Journal of Stem Cells
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• v.17 no.2
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• pp.130-140
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• 2024

Cardiac organoids have emerged as invaluable tools for assessing the impact of diverse substances on heart function. This report introduces guidelines for general requirements for manufacturing cardiac organoids and conducting cardiac organoid-based assays, encompassing protocols, analytical methodologies, and ethical considerations. In the quest to employ recently developed three-dimensional cardiac organoid models as substitutes for animal testing, it becomes imperative to establish robust criteria for evaluating organoid quality and conducting toxicity assessments. This guideline addresses this need, catering to regulatory requirements, and describes common standards for organoid quality and toxicity assessment methodologies, commensurate with current technological capabilities. While acknowledging the dynamic nature of technological progress and the potential for future comparative studies, this guideline serves as a foundational framework. It offers a comprehensive approach to standardized cardiac organoid testing, ensuring scientific rigor, reproducibility, and ethical integrity in investigations of cardiotoxicity, particularly through the utilization of human pluripotent stem cell-derived cardiac organoids.

• Development and Reproduction
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• v.14 no.4
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• pp.243-251
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• 2010

ESRRB (Estrogen related receptor $\beta$) is an orphan receptor, and have a role on maintaining the undifferentiated state and self-renewal of pluripotent stem cell as a transcription factor which regulates the expression of OCT4 and NANOG genes. Also, Feng et al. (2009) reported that Esrrb, Oct4 and Sox2 could induce pluripotent stem cell from somatic cells. The aim of the present study was to develop the direct delivery system of human ESRRB protein into human amniotic fluid-derived stem cells (AFSCs) and to analyze the effect of ESRRB on the regulation of pluripotency-related genes. Human ESRRB has three isoforms arisen by alternative splicing. We cloned short-form ESRRB and made a fusion protein of ESRRB and R7 for an efficient protein transfer to cell. R7 as cell-penetrating peptide(CPP) can help to transfer ESRRB into cells. R7-ESRRB-His6 protein was observed in the cytoplasm and nuclei within 5 hours after treatment. Also, we could observe R7-ESRRB-His6 protein only in the nuclei within 24 hours. Realtime PCR showed that ESRRB increased expression of OCT4 and NANOG as well as SOX2 gene. Therefore, we demonstrated that R7-ESRRB-His6 proteins were efficiently transferred into the nuclei of AFSCs and work well as a possible transcription factor.

• Reproductive and Developmental Biology
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• v.39 no.3
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• pp.89-95
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• 2015

The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.

• 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.

• 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.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• 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.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.281-292
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• 2023

Background and Objectives: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. Methods and Results: We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring-mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. Conclusions: To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

• BMB Reports
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• v.39 no.4
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• pp.426-431
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• 2006

Embryonic stem cells (ESCs), representing a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics, are capable of spontaneous differentiation into cardiomyocytes. The present study sought to define the kinetic characterization of lactate dehydrogenase (LDH) and creatine kinase (CK) of ESC- and neonatal-derived cardiomyocytes. Spontaneously differentiated cardiomyocytes from embryoid bodies (EBs) derived from mouse ESC line (Royan B1) and neonatal cardiomyocytes were dispersed in a buffer solution. Enzymes were extracted by sonication and centrifugation for kinetic evaluation of LDH and CK with spectrophotometric methods. While a comparison between the kinetic properties of the LDH and CK of both groups revealed not only different Michaelis constants and optimum temperatures for LDH but also different Michaelis constants and optimum pH for CK, the pH profile of LDH and optimum temperature of CK were similar. In defining some kinetic properties of cardiac metabolic enzymes of ESC-derived cardiomyocytes, our results are expected to further facilitate the use of ESCs as an experimental model.