• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.449-455
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• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• Molecules and Cells
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• v.41 no.11
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• pp.971-978
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• 2018

The stem cell factor (SCF)/c-KIT axis plays an important role in the hematopoietic differentiation of human pluripotent stem cells (hPSCs), but its regulatory mechanisms involving microRNAs (miRs) are not fully elucidated. Here, we demonstrated that supplementation with SCF increases the hematopoietic differentiation of hPSCs via the interaction with its receptor tyrosine kinase c-KIT, which is modulated by miR-221 and miR-222. c-KIT is comparably expressed in undifferentiated human embryonic and induced pluripotent stem cells. The inhibition of SCF signaling via treatment with a c-KIT antagonist (imatinib) during hPSC-derived hematopoiesis resulted in reductions in the yield and multi-lineage potential of hematopoietic progenitors. We found that the transcript levels of miR-221 and miR-222 targeting c-KIT were significantly lower in the pluripotent state than they were in terminally differentiated somatic cells. Furthermore, suppression of miR-221 and miR-222 in undifferentiated hPSC cultures induced more hematopoiesis by increasing c-KIT expression. Collectively, our data implied that the modulation of c-KIT by miRs may provide further potential strategies to expedite the generation of functional blood cells for therapeutic approaches and the study of the cellular machinery related to hematologic malignant diseases such as leukemia.

• 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.

• International Journal of Stem Cells
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• v.17 no.2
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• pp.194-203
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• 2024

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time, with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2~4 and 2~3 hours after starvation, respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs, focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types, including undifferentiated PSCs, differentiated cells, and cells undergoing cellular reprogramming, and addresses critical issues, such as differences in basal metabolic levels and sensitivity to normalization, providing valuable insights into cellular energetics.

• 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.

• Development and Reproduction
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• v.27 no.2
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• pp.57-65
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• 2023

Kidney disease affects a significant portion of the global population, yet effective therapies are lacking despite advancements in identifying genetic causes. This limitation can be attributed to the absence of adequate in vitro models that accurately mimic human kidney disease, hindering targeted therapeutic development. However, the emergence of human induced pluripotent stem cells (PSCs) and the development of organoids using them have opened up a way to model kidney development and disease in humans, as well as validate the effects of new drugs. To fully leverage their capabilities in these fields, it is crucial for kidney organoids to closely resemble the structure and functionality of adult human kidneys. In this review, we aim to discuss the potential of using human PSCs or adult kidney stem cell-derived kidney organoids to model genetic kidney disease and renal cancer.

• The Journal of the Korean dental association
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• v.55 no.12
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• pp.828-840
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• 2017

In recent years, many researchers and clinicians found interest in regenerative medicine using induced pluripotent stem cells (iPSCs) with biomaterials due to their pluripotency, which is able to differentiate into any type of cells without human embryo, which of use is ethically controversial. However, there are limitations to make iPSCs from adult somatic cells due to their low stemness and donor site morbidity. Recently, to overcome above drawbacks, dental tissue-derived iPSCs have been highlighted as a type of alternative sources for their high stemness, easy gathering, and their complex (ectomesenchymal) origin, which easily differentiate them to various cell types for nerve, vessel, and other dental tissue regeneration. In other part, utilizing biomaterials for regenerative medicine using cell is recently highlighted because they can modulate cell adhesion, proliferation and (de)differentiation. Therefore, this paper will convey the overview of advantages and drawbacks of dental tissue-derived iPSCs and their future application with biomaterials.

• 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.

• BMB Reports
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• v.52 no.12
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• pp.689-694
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• 2019

Ethical and safety issues have rendered mesenchymal stem cells (MSCs) popular candidates in regenerative medicine, but their therapeutic capacity is lower than that of induced pluripotent stem cells (iPSCs). This study compared original, dental tissue-derived MSCs with re-differentiated MSCs from iPSCs (iPS-MSCs). CD marker expression in iPS-MSCs was similar to original MSCs. iPS-MSCs expressed higher in pluripotent genes, but lower levels in mesodermal genes than MSCs. In addition, iPS-MSCs did not form teratomas. All iPSCs carried mtDNA mutations; some shared with original MSCs and others not previously detected therein. Shared mutations were synonymous, while novel mutations were non-synonymous or located on RNA-encoding genes. iPS-MSCs also harbored mtDNA mutations transmitted from iPSCs. Selected iPS-MSCs displayed lower mitochondrial respiration than original MSCs. In conclusion, screening for mtDNA mutations in iPSC lines for iPS-MSCs can identify mutation-free cell lines for therapeutic applications.

• International Journal of Stem Cells
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• v.17 no.1
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• pp.59-69
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• 2024

Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.