Publications by authors named "Hyung-Min Chung"

178 Publications

In vivo tracking of C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry.

Sci Rep 2021 Jan 14;11(1):1360. Epub 2021 Jan 14.

Department of Stem Cell Biology, School of Medicine, Konkuk University, 120 Neungdong-Ro, Gwangjin-Gu, 05029, Republic of Korea.

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.
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http://dx.doi.org/10.1038/s41598-020-80416-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809063PMC
January 2021

Anti-Inflammatory Effects of M-MSCs in DNCB-Induced Atopic Dermatitis Mice.

Biomedicines 2020 Oct 21;8(10). Epub 2020 Oct 21.

Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 05029, Korea.

Atopic dermatitis (AD) is an inflammatory skin disease caused by an imbalance between Th1 and Th2 cells. AD patients suffer from pruritus, excessive dryness, red or inflamed skin, and complications such as sleep disturbances and depression. Although there are currently many AD treatments available there are insufficient data on their long-term stability and comparative effects. Moreover, they have limitations due to various side effects. Multipotent mesenchymal stem cells (M-MSCs) might have potential for next-generation AD therapies. MSCs are capable of immune function regulation and local inflammatory response inhibition. M-MSCs, derived from human embryonic stem cells (hESC), additionally have a stable supply. In L507 antibody array, M-MSCs generally showed similar tendencies to bone marrow-derived mesenchymal stem cells (BM-MSCs), although the immunoregulatory function of M-MSCs seemed to be superior to BM-MSCs. Based on the characteristics of M-MSCs on immunoregulatory functions, we tested a M-MSC conditioned media concentrate (MCMC) in mice with AD lesions on their dorsal skin. MCMC significantly decreased RNA expression levels of inflammatory cytokines in the mouse dorsal skin. It also suppressed serum IgE levels. In addition, significant histopathologic alleviation was identified. In conclusion, secretions of M-MSCs have the potential to effectively improve AD-related inflammatory lesions. M-MSCs showed potential for use in next-generation AD treatment.
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http://dx.doi.org/10.3390/biomedicines8100439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589030PMC
October 2020

Therapeutic Efficacy of Human Embryonic Stem Cell-Derived Multipotent Stem/Stromal Cells in Diabetic Detrusor Underactivity: A Preclinical Study.

J Clin Med 2020 Sep 3;9(9). Epub 2020 Sep 3.

Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea.

Mesenchymal stem/stromal cell (MSC) therapy is a promising approach for treatment of as yet incurable detrusor underactivity (DUA), which is characterized by decreased detrusor contraction strength and/or duration, leading to prolonged bladder emptying. In the present study, we demonstrated the therapeutic potential of human embryonic stem cell (ESC)-derived multipotent MSCs (M-MSCs) in a diabetic rat model of DUA. Diabetes mellitus (DM) was induced by intraperitoneal injection of streptozotocin (STZ) (50 mg/kg) into 8-week-old female Sprague-Dawley rats. Three weeks later, various doses of M-MSCs (0.25, 0.5, and 1 × 10 cells) or an equivalent volume of PBS were injected into the outer layer of the bladder. Awake cystometry, organ bath, histological, and gene expression analyses were evaluated 1 week (short-term) or 2 and 4 weeks (long-term) after M-MSC transplantation. STZ-induced diabetic rats developed DUA, including phenotypes with significantly longer micturition intervals, increased residual urine amounts and bladder capacity, decreased micturition pressure on awake cystometry, and contractile responses to various stimuli in organ bath studies. Muscle degeneration, mast cell infiltration, fibrosis, and apoptosis were present in the bladders of DM animals. A single local transplantation of M-MSCs ameliorated DUA bladder pathology, including functional changes and histological evaluation, and caused few adverse outcomes. Immunostaining and gene expression analysis revealed that the transplanted M-MSCs supported myogenic restoration primarily by engrafting into bladder tissue via pericytes, and subsequently exerting paracrine effects to prevent apoptotic cell death in bladder tissue. The therapeutic efficacy of M-MSCs was superior to that of human umbilical cord-derived MSCs at the early time point (1 week). However, the difference in efficacy between M-MSCs and human umbilical cord-derived MSCs was statistically insignificant at the later time points (2 and 4 weeks). Collectively, the present study provides the first evidence for improved therapeutic efficacy of a human ESC derivative in a preclinical model of DM-associated DUA.
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http://dx.doi.org/10.3390/jcm9092853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563486PMC
September 2020

Glutathione dynamics determine the therapeutic efficacy of mesenchymal stem cells for graft-versus-host disease via CREB1-NRF2 pathway.

Sci Adv 2020 Apr 15;6(16):eaba1334. Epub 2020 Apr 15.

Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea.

Glutathione (GSH), the most abundant nonprotein thiol functioning as an antioxidant, plays critical roles in maintaining the core functions of mesenchymal stem cells (MSCs), which are used as a cellular immunotherapy for graft-versus-host disease (GVHD). However, the role of GSH dynamics in MSCs remains elusive. Genome-wide gene expression profiling and high-throughput live-cell imaging assays revealed that CREB1 enforced the GSH-recovering capacity (GRC) of MSCs through NRF2 by directly up-regulating NRF2 target genes responsible for GSH synthesis and redox cycling. MSCs with enhanced GSH levels and GRC mediated by CREB1-NRF2 have improved self-renewal, migratory, anti-inflammatory, and T cell suppression capacities. Administration of MSCs overexpressing CREB1-NRF2 target genes alleviated GVHD in a humanized mouse model, resulting in improved survival, decreased weight loss, and reduced histopathologic damages in GVHD target organs. Collectively, these findings demonstrate the molecular and functional importance of the CREB1-NRF2 pathway in maintaining MSC GSH dynamics, determining therapeutic outcomes for GVHD treatment.
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http://dx.doi.org/10.1126/sciadv.aba1334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239701PMC
April 2020

Application of co-culture technology of epithelial type cells and mesenchymal type cells using nanopatterned structures.

PLoS One 2020 11;15(5):e0232899. Epub 2020 May 11.

Research Institute, T&R Biofab Co. Ltd, Siheung, Republic of Korea.

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232899PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213697PMC
August 2020

Development and evaluation of next-generation cardiotoxicity assay based on embryonic stem cell-derived cardiomyocytes.

BMB Rep 2020 Aug;53(8):437-441

Department of Medicine, School of Medicine, Konkuk University, Seoul 05029, Korea.

In accordance with requirements of the ICH S7B safety pharmacology guidelines, numerous next-generation cardiotoxicity studies using human stem cell-derived cardiomyocytes (CMs) are being conducted globally. Although several stem cell-derived CMs are being developed for commercialization, there is insufficient research to verify if these CMs can replace animal experiments. In this study, in vitro high-efficiency CMs derived from human embryonic stem cells (hESC-CMs) were compared with Sprague-Dawley rats as in vivo experimental animals, and primary cultured in vitro rat-CMs for cardiotoxicity tests. In vivo rats were administrated with two consecutive injections of 100 mg/kg isoproterenol, 15 mg/kg doxorubicin, or 100 mg/kg nifedipine, while in vitro rat-CMs and hESC-CMs were treated with 5 μM isoproterenol, 5 μM doxorubicin, and 50 μM nifedipine. We have verified the equivalence of hESC-CMs assessments over various molecular biological markers, morphological analysis. Also, we have identified the advantages of hESC-CMs, which can distinguish between species variability, over electrophysiological analysis of ion channels against cardiac damage. Our findings demonstrate the possibility and advantage of high-efficiency hESC-CMs as next-generation cardiotoxicity assessment. [BMB Reports 2020; 53(8): 437-441].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473479PMC
August 2020

Effect of Cell Labeling on the Function of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Int J Stem Cells 2020 Jul;13(2):287-294

Department of Human and Environmental Toxicology, University of Science and Technology, Daejeon, Korea.

Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3'-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca influx by L-type Ca channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.
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http://dx.doi.org/10.15283/ijsc19138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378900PMC
July 2020

Comparative analysis of the mitochondrial morphology, energy metabolism, and gene expression signatures in three types of blastocyst-derived stem cells.

Redox Biol 2020 02 20;30:101437. Epub 2020 Jan 20.

Department of Stem Cell and Regenerative Biotechnology, Konkuk Institute of Technology, Konkuk University, Seoul, Republic of Korea. Electronic address:

Pre-implantation mouse blastocyst-derived stem cells, namely embryonic stem cells (ESCs), trophoblast stem cells (TSCs), and extraembryonic endoderm (XEN) cells, have their own characteristics and lineage specificity. So far, several studies have attempted to identify these three stem cell types based on genetic markers, morphologies, and factors involved in maintaining cell self-renewal. In this study, we focused on characterizing the three stem cell types derived from mouse blastocysts by observing cellular organelles, especially the mitochondria, and analyzing how mitochondrial dynamics relates to the energy metabolism in each cell type. Our study revealed that XEN cells have distinct mitochondrial morphology and energy metabolism compared with that in ESCs and TSCs. In addition, by analyzing the energy metabolism (oxygen consumption and extracellular acidification rates), we demonstrated that differences in the mitochondria affect the cellular metabolism in the stem cells. RNA sequencing analysis showed that although ESCs are developmentally closer to XEN cells in origin, their gene expression pattern is relatively closer to that of TSCs. Notably, mitochondria-, mitochondrial metabolism-, transport/secretory action-associated genes were differentially expressed in XEN cells compared with that in ESCs and TSCs, and this feature corresponds with the morphology of the cells.
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http://dx.doi.org/10.1016/j.redox.2020.101437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992993PMC
February 2020

Epigenetic priming by Dot1l in lymphatic endothelial progenitors ensures normal lymphatic development and function.

Cell Death Dis 2020 01 6;11(1):14. Epub 2020 Jan 6.

Department of Stem Cell & Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Gwangjin-gu, 05029, Republic of Korea.

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood-lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.
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http://dx.doi.org/10.1038/s41419-019-2201-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944698PMC
January 2020

Ascorbic Acid 2-Glucoside Stably Promotes the Primitiveness of Embryonic and Mesenchymal Stem Cells Through Ten-Eleven Translocation- and cAMP-Responsive Element-Binding Protein-1-Dependent Mechanisms.

Antioxid Redox Signal 2020 01;32(1):35-59

Department of Biomedical Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

The naive or primitive states of stem cells (SCs) residing in specific niches are unstable and difficult to preserve . Vitamin C (VitC), in addition to suppressing oxygen radicals, exerts pleiotropic effects to preserve the core functions of SCs. However, this compound is labile and readily oxidized, resulting in cellular toxicity and preventing its reliable application in this context. We found that a VitC derivative, ascorbic acid 2-glucoside (AA2G), stably maintains the naive pluripotency of murine embryonic SCs (mESCs) and the primitiveness of human mesenchymal SCs (hMSCs) without cellular toxicity. The beneficial effects of AA2G and related molecular mechanisms were evaluated in mESCs, induced pluripotent-SCs (iPSCs), and hMSCs. AA2G was stable in aqueous solution and barely induced cellular toxicity in cultured SCs, unlike VitC. AA2G supplementation recapitulated the well-known effects of VitC, including induction of ten-eleven translocation-dependent DNA demethylation in mESCs and suppression of p53 during generation of murine iPSCs. Furthermore, supplementation of hMSCs with AA2G improved therapeutic outcomes in an asthma mouse model by promoting their self-renewal, engraftment, and anti-inflammatory properties. Particularly, activation of the cAMP-responsive element-binding protein-1 (CREB1) pathway contributed to the ability of AA2G to maintain naive pluripotency of mESCs and functionality of hMSCs. Given its long-lasting effects and low cellular toxicity, AA2G supplementation is useful to support the naive pluripotency of mESCs and the primitiveness of hMSCs, affecting their developmental potency and therapeutic efficacy. Furthermore, we demonstrate the significance of the CREB1 pathway in the mechanism of action of AA2G.
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http://dx.doi.org/10.1089/ars.2019.7743DOI Listing
January 2020

Reproducible hindlimb ischemia model based on photochemically induced thrombosis to evaluate angiogenic effects.

Microvasc Res 2019 11 18;126:103912. Epub 2019 Aug 18.

Research Group for Biomimetic Advanced Technology, Korea Institute of Toxicology, Daejeon, Republic of Korea; Department of Human and Environmental Toxicology, University of Science and Technology, Daejeon, Republic of Korea. Electronic address:

Critical limb ischemia is one of the most common types of peripheral arterial disease. Preclinical development of ischemia therapeutics relies on the availability of a relevant and reproducible in vivo disease model. Thus, establishing appropriate animal disease models is essential for the development of new therapeutic strategies. Currently, the most commonly employed model of hindlimb ischemia is the surgical induction method with ligation of the femoral artery and its branches after skin incision. However, the efficiency of the method is highly variable depending on the availability of skilled technicians. In addition, after surgical procedures, animals can quickly and spontaneously recover from damage, limiting observations of the therapeutic effect of potential agents. The aim of this study was to develop a hindlimb ischemia mouse model with similarities to human ischemic disease. To that end, a photochemical reaction was used to induce thrombosis in the hindlimb. After the photochemical reaction was induced by light irradiation, thrombotic plugs and adjacent red blood cell stasis were observed in hindlimb vessels in the light-irradiated zone. Additionally, the photochemically induced thrombosis maintained the ischemic condition and did not cause notable side effects in mice.
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http://dx.doi.org/10.1016/j.mvr.2019.103912DOI Listing
November 2019

Cellular organization of three germ layer cells on different types of noncovalent functionalized graphene substrates.

Mater Sci Eng C Mater Biol Appl 2019 Oct 20;103:109729. Epub 2019 May 20.

Department of Energy Engineering, Konkuk University, Seoul 05029, Republic of Korea; Division of Stem Cell Research, T&R Biofab Co. Ltd, Seongnam-si 13494, Republic of Korea. Electronic address:

Graphene and its derivatives have seen a rapid rise in interest as promising biomaterials especially in the field of tissue engineering, regenerative medicine, and cell biology of late. Despite its proven potential in numerous biological applications, information regarding the relationship between the different forms of graphene and cell lineages is still lacking partly due to its topical emergence in cellular studies. Herein, we explore the biocompatibility of four types of graphene substrates (chemical vapor deposition grown graphene, mechanically exfoliated graphene, chemically exfoliated graphene oxide, and reduced graphene oxide) with three types of somatic cells (keratinocytes, hepatocytes, endothelial cells) derived from the three germ layers in relation to cell adhesion, proliferation, morphology, and gene expression. The results revealed exceptional cell adhesion for all tested groups but enhanced proliferation and cytoskeletal interconnectivity in graphene oxide and reduced graphene oxide substrates. We were unable to detect any adverse effects in gene expression and survivability during a week of culture. We further show topographic changes to graphene substrates under fetal bovine serum adsorption to better illustrate the actual microenvironment of inhabitant cells. This study highlights the extraordinary synergy between graphene and somatic cells, suggesting the discretionary use of extracellular matrix components for in vitro cultivation.
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http://dx.doi.org/10.1016/j.msec.2019.05.014DOI Listing
October 2019

Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction.

Nat Commun 2019 07 16;10(1):3123. Epub 2019 Jul 16.

Department of Medical Life Science, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 137701, Republic of Korea.

Since both myocardium and vasculature in the heart are excessively damaged following myocardial infarction (MI), therapeutic strategies for treating MI hearts should concurrently target both so as to achieve true cardiac repair. Here we demonstrate a concomitant method that exploits the advantages of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and human mesenchymal stem cell-loaded patch (hMSC-PA) to amplify cardiac repair in a rat MI model. Epicardially implanted hMSC-PA provide a complimentary microenvironment which enhances vascular regeneration through prolonged secretion of paracrine factors, but more importantly it significantly improves the retention and engraftment of intramyocardially injected hiPSC-CMs which ultimately restore the cardiac function. Notably, the majority of injected hiPSC-CMs display adult CMs like morphology suggesting that the secretomic milieu of hMSC-PA constitutes pleiotropic effects in vivo. We provide compelling evidence that this dual approach can be a promising means to enhance cardiac repair on MI hearts.
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http://dx.doi.org/10.1038/s41467-019-11091-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635499PMC
July 2019

The Effect of Hexanoyl Glycol Chitosan on the Proliferation of Human Mesenchymal Stem Cells.

Polymers (Basel) 2018 Jul 30;10(8). Epub 2018 Jul 30.

Department of Medicine, School of Medicine, Konkuk University, Seoul 05029, Korea.

Adipose-derived mesenchymal stem cells (AD-MSCs) have been studied as desirable cell sources for regenerative medicine and therapeutic application. However, it has still remained a challenge to obtain enough adequate and healthy cells in large quantities. To overcome this limitation, various biomaterials have been used to promote expansion of MSCs in vitro. Recently, hexanoyl glycol chitosan (HGC) was introduced as a new biomaterial for various biomedical applications, in particular 3D cell culture, because of its biodegradability, biocompatibility, and other promising biofunctional properties. In this study, the effect of HGC on the proliferation of AD-MSCs was examined in vitro and its synergistic effect with basic fibroblast growth factor (bFGF), which has been widely used to promote proliferation of cells, was evaluated. We found that the presence of HGC increased the proliferative capacity of AD-MSCs during long-term culture, even at low concentrations of bFGF. Furthermore, it suppressed the expression of senescence-related genes and improved the mitochondrial functionality. Taken all together, these findings suggest that the HGC demonstrate a potential for sustained growth of AD-MSCs in vitro.
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http://dx.doi.org/10.3390/polym10080839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404012PMC
July 2018

Functional Equivalency in Human Somatic Cell Nuclear Transfer-Derived Endothelial Cells.

Stem Cells 2019 05 22;37(5):623-630. Epub 2019 Feb 22.

Department of Stem Cell Biology, Konkuk University, School of Medicine, Seoul, Republic of Korea.

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.
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http://dx.doi.org/10.1002/stem.2986DOI Listing
May 2019

Tristetraprolin-mediated hexokinase 2 expression regulation contributes to glycolysis in cancer cells.

Mol Biol Cell 2019 03 16;30(5):542-553. Epub 2019 Jan 16.

Department of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea.

Hexokinase 2 (HK2) catalyzes the first step of glycolysis and is up-regulated in cancer cells. The mechanism has not been fully elucidated. Tristetraprolin (TTP) is an AU-rich element (ARE)-binding protein that inhibits the expression of ARE-containing genes by enhancing mRNA degradation. TTP expression is down-regulated in cancer cells. We demonstrated that TTP is critical for down-regulation of HK2 expression in cancer cells. HK2 mRNA contains an ARE within its 3'-UTR. TTP binds to HK2 3'-UTR and enhances degradation of HK2 mRNA. TTP overexpression decreased HK2 expression and suppressed the glycolytic capacity of cancer cells, measured as glucose uptake and production of glucose-6-phosphate, pyruvate, and lactate. TTP overexpression reduced both the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cancer cells. Ectopic expression of HK2 in cancer cells attenuated the reduction in glycolytic capacity, ECAR, and OCR from TTP. Taken together, these findings suggest that TTP acts as a negative regulator of HK2 expression and glucose metabolism in cancer cells.
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http://dx.doi.org/10.1091/mbc.E18-09-0606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589696PMC
March 2019

Longitudinal intravital imaging of transplanted mesenchymal stem cells elucidates their functional integration and therapeutic potency in an animal model of interstitial cystitis/bladder pain syndrome.

Theranostics 2018 9;8(20):5610-5624. Epub 2018 Nov 9.

Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

Mesenchymal stem cell (MSC) therapy may be a novel approach to improve interstitial cystitis/bladder pain syndrome (IC/BPS), an intractable disease characterized by severe pelvic pain and urinary frequency. Unfortunately, the properties of transplanted stem cells have not been directly analyzed , which hampers elucidation of the therapeutic mechanisms of these cells and optimization of transplantation protocols. Here, we monitored the behaviors of multipotent stem cells (M-MSCs) derived from human embryonic stem cells (hESCs) in real time using a novel combination of confocal endoscopic and microscopic imaging and demonstrated their improved therapeutic potency in a chronic IC/BPS animal model. Ten-week-old female Sprague-Dawley rats were instilled with 10 mg of protamine sulfate followed by 750 μg of lipopolysaccharide weekly for 5 weeks. The sham group was instilled with phosphate-buffered saline (PBS). Thereafter, the indicated dose (0.1, 0.25, 0.5, and 1×10 cells) of M-MSCs or PBS was injected once into the outer layer of the bladder. The distribution, perivascular integration, and therapeutic effects of M-MSCs were monitored by endoscopic and confocal microscopic imaging, awake cystometry, and histological and gene expression analyses. A novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, together with immunofluorescence analysis of bladder tissues, demonstrated that transplanted M-MSCs engrafted following differentiation into multiple cell types and gradually integrated into a perivascular-like structure until 30 days after transplantation. The beneficial effects of transplanted M-MSCs on bladder voiding function and the pathological characteristics of the bladder were efficient and long-lasting due to the stable engraftment of these cells. This longitudinal bioimaging study of transplanted hESC-derived M-MSCs in living animals reveals their long-term functional integration, which underlies the improved therapeutic effects of these cells on IC/BPS.
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http://dx.doi.org/10.7150/thno.27559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276303PMC
September 2019

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts.

Anim Cells Syst (Seoul) 2018 15;22(2):132-139. Epub 2018 Mar 15.

Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Korea.

Generation of induced pluripotent stem cells (iPSCs) by defined factors (, , , and ) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (, , , and ) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (/, /, /) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.
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http://dx.doi.org/10.1080/19768354.2018.1451367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138300PMC
March 2018

Reconstituting Human Cutaneous Regeneration in Humanized Mice under Endothelial Cell Therapy.

J Invest Dermatol 2019 03 28;139(3):692-701. Epub 2018 Oct 28.

Department of Medicine, School of Medicine, Konkuk University, Seoul, Republic of Korea. Electronic address:

Much of our understanding of human biology and the function of mammalian cells in tissue regeneration have been derived from mechanistically and genetically manipulated rodent models. However, current models examining epidermal wound repair fail to address both the cross-species mechanistic and immunogenic differences simultaneously. Herein, we describe a multifaceted approach intended to better recapitulate human skin recovery in rodent models. First, immunodeficient NOD.Cg-PrkdcIl2rg/SzJ mice were intravenously inoculated with human hematopoietic stem cells to become, in essence, humanized, and capable of initiating an adaptive immune response. Next, a chimney-shaped mechanical device was implanted onto the excisional wound site to prevent healing by primary intention (contraction) and expedite cell transplantation. Subsequently, cell therapy was administered by transplanting cord blood-derived endothelial progenitor cells or human pluripotent stem cell-derived endothelial cells into the wound site to examine the regeneration process at a histological level. This study demonstrates human cutaneous repair in a murine model by addressing both the mechanistic and immunogenic differences in the epidermis. We further show human leukocyte recruitment in damaged tissue and improved healing by secondary intention in the transplanted groups, highlighting the need for useful preclinical animal models to better understand leukocyte function in human (tissue repair and) regeneration.
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http://dx.doi.org/10.1016/j.jid.2018.08.031DOI Listing
March 2019

The Pharmacological Inhibition of ERK5 Enhances Apoptosis in Acute Myeloid Leukemia Cells.

Int J Stem Cells 2018 Nov;11(2):227-234

Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Korea.

Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Extracellular signal-regulated kinase 5 (ERK5) plays a novel role in chemoresistance in some cancer cells and this pathway is a central mediator of cell survival and apoptotic regulation. The aim of this study was to investigate the effect of ERK5 inhibitor, XMD8-92, on proliferation and apoptosis in AML cell lines. Findings showed that XMD8-92 inhibited the activation of ERK5 by G-CSF and decreased the expression of c-Myc and Cyclin D1. The treatment of XMD8-92 reduced the phosphorylation of ERK5 leading to a distinct inhibition of cell proliferation and increased apoptosis in Kasumi-1 and HL-60 cells. Taken together, our study suggests that the inhibition of ERK5 by XMD8-92 can trigger apoptosis and inhibit proliferation in AMLs. Therefore, the inhibition of ERK5 may be an effective adjuvant in AML chemotherapy.
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http://dx.doi.org/10.15283/ijsc18053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6285287PMC
November 2018

Mesenchymal stem cell-driven activatable photosensitizers for precision photodynamic oncotherapy.

Biomaterials 2018 12 28;187:18-26. Epub 2018 Sep 28.

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, South Korea. Electronic address:

Precise targeting with minimal side effects is of particular interest for personalized medicine, although it remains a challenge. Herein, we demonstrate precision photodynamic therapy (PDT) utilizing human mesenchymal stem cells (MSCs) as cellular vehicles to deliver a new activatable photosensitizer (PcS). In vivo real-time optical imaging tests indicated that PcS-loaded MSCs possess excellent tumor-homing properties. More importantly, dye transfer assays confirm that MSCs precisely transfer PcS into human colon cancer cells (HCT116) via the "bystander effect." Upon localized light irradiation, the growth of intraperitoneal xenograft tumors was significantly inhibited by the photodynamic effect. These findings represent a promising strategy for precise oncotherapy.
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http://dx.doi.org/10.1016/j.biomaterials.2018.09.041DOI Listing
December 2018

Safety and Optimization of Metabolic Labeling of Endothelial Progenitor Cells for Tracking.

Sci Rep 2018 09 4;8(1):13212. Epub 2018 Sep 4.

Predictive Model Research Center, Korea Institute of Toxicology, Daejeon, Korea.

Metabolic labeling is one of the most powerful methods to label the live cell for in vitro and in vivo tracking. However, the cellular mechanisms by modified glycosylation due to metabolic agents are not fully understood. Therefore, metabolic labeling has not yet been widely used in EPC tracking and labeling. In this study, cell functional properties such as proliferation, migration and permeability and gene expression patterns of metabolic labeling agent-treated hUCB-EPCs were analyzed to demonstrate cellular effects of metabolic labeling agents. As the results, 10 μM Ac4ManNAz treatment had no effects on cellular function or gene regulations, however, higher concentration of Ac4ManNAz (>20 μM) led to the inhibition of functional properties (proliferation rate, viability and rate of endocytosis) and down-regulation of genes related to cell adhesion, PI3K/AKT, FGF and EGFR signaling pathways. Interestingly, the new blood vessel formation and angiogenic potential of hUCB-EPCs were not affected by Ac4ManNAz concentration. Based on our results, we suggest 10 μM as the optimal concentration of Ac4ManNAz for in vivo hUCB-EPC labeling and tracking. Additionally, we expect that our approach can be used for understanding the efficacy and safety of stem cell-based therapy in vivo.
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http://dx.doi.org/10.1038/s41598-018-31594-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123424PMC
September 2018

Improved Transfection Efficiency and Metabolic Activity in Human Embryonic Stem Cell Using Non-Enzymatic Method.

Int J Stem Cells 2018 Nov;11(2):149-156

Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Korea.

Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.
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http://dx.doi.org/10.15283/ijsc18037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6285293PMC
November 2018

Effect of Graphene Nanoribbons (TexasPEG) on locomotor function recovery in a rat model of lumbar spinal cord transection.

Neural Regen Res 2018 Aug;13(8):1440-1446

HEAVEN/GEMINI International Collaborative Group, Turin, Italy.

A sharply transected spinal cord has been shown to be fused under the accelerating influence of membrane fusogens such as polyethylene glycol (PEG) (GEMINI protocol). Previous work provided evidence that this is in fact possible. Other fusogens might improve current results. In this study, we aimed to assess the effects of PEGylated graphene nanoribons (PEG-GNR, and called "TexasPEG" when prepared as 1wt% dispersion in PEG600) versus placebo (saline) on locomotor function recovery and cellular level in a rat model of spinal cord transection at lumbar segment 1 (L) level. In vivo and in vitro experiments (n = 10 per experiment) were designed. In the in vivo experiment, all rats were submitted to full spinal cord transection at L level. Five weeks later, behavioral assessment was performed using the Basso Beattie Bresnahan (BBB) locomotor rating scale. Immunohistochemical staining with neuron marker neurofilament 200 (NF200) antibody and astrocytic scar marker glial fibrillary acidic protein (GFAP) was also performed in the injured spinal cord. In the in vitro experiment, the effects of TexasPEG application for 72 hours on the neurite outgrowth of SH-SY5Y cells were observed under the inverted microscope. Results of both in vivo and in vitro experiments suggest that TexasPEG reduces the formation of glial scars, promotes the regeneration of neurites, and thereby contributes to the recovery of locomotor function of a rat model of spinal cord transfection.
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http://dx.doi.org/10.4103/1673-5374.235301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108198PMC
August 2018

Two-Step Generation of Oligodendrocyte Progenitor Cells From Mouse Fibroblasts for Spinal Cord Injury.

Front Cell Neurosci 2018 25;12:198. Epub 2018 Jul 25.

Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, South Korea.

Oligodendrocyte progenitor cells (OPCs) are attracting attention as the ideal cell therapy for spinal cord injury (SCI). Recently, advanced reprogramming and differentiation techniques have made it possible to generate therapeutic cells for treating SCI. In the present study, we used directly-induced neural stem cells (DNSCs) from fibroblasts to establish OPCs (DN-OPCs) capable of proliferation and confirmed their OPC-specific characteristics. Also, we evaluated the effect of transplanted DN-OPCs on SCI in rats. The DN-OPCs exhibited an OPC-specific phenotype and electrophysiological function and could be differentiated into oligodendrocytes. In the SCI model, transplanted DN-OPCs improved behavior recovery, and showed engraftment into the host spinal cord with expression of myelin basic protein. These results suggest that DN-OPCs could be a new source of potentially useful cells for treating SCI.
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http://dx.doi.org/10.3389/fncel.2018.00198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070016PMC
July 2018

The Bromodomain Inhibitor JQ1 Enhances the Responses to All- Retinoic Acid in HL-60 and MV4-11 Leukemia Cells.

Int J Stem Cells 2018 May;11(1):131-140

Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Korea.

All- retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML). However, ATRA-based treatment is not effective in other subtypes of AML. In non-APL AML, ATRA signaling pathway is impaired or downmodulated, and consequently fails to respond to pharmacological doses of ATRA. Therefore, complementary treatment strategies are needed to improve ATRA responsiveness in non-APL AML. In this study, we investigated the combined effect of ATRA and bromodomain inhibitor JQ1, proven to have potent anti-cancer activity mainly through inhibition of c-Myc. We showed that the combination of ATRA with JQ1 synergistically inhibited proliferation of AML cells. The synergistic growth inhibition was resulted from differentiation or apoptosis depending on the kind of AML cells. Concomitantly, the combined treatment of ATRA and JQ1 caused greater depletion of c-Myc and hTERT expression than each agent alone in AML cells. Taken together, these findings support the rationale for the use of the combination of ATRA and JQ1 as a therapeutic strategy for the treatment of AML.
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http://dx.doi.org/10.15283/ijsc18021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984067PMC
May 2018

Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells.

Epigenetics 2018 3;13(4):343-351. Epub 2018 May 3.

a Department of Stem Cell Biology, School of Medicine , Konkuk University , Seoul 05029 , Korea.

Genomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.
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http://dx.doi.org/10.1080/15592294.2018.1460033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140819PMC
February 2019

The Therapeutic Effect of Human Embryonic Stem Cell-Derived Multipotent Mesenchymal Stem Cells on Chemical-Induced Cystitis in Rats.

Int Neurourol J 2018 Jan 31;22(Suppl 1):S34-45. Epub 2018 Jan 31.

Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

Purpose: To evaluate the therapeutic effect of human embryonic stem cell (hESC)-derived multipotent mesenchymal stem cells (M-MSCs) on ketamine-induced cystitis (KC) in rats.

Methods: To induce KC, 10-week-old female rats were injected with 25-mg/kg ketamine hydrochloride twice weekly for 12 weeks. In the sham group, phosphate buffered saline (PBS) was injected instead of ketamine. One week after the final injection of ketamine, the indicated doses (0.25, 0.5, and 1×10 cells) of M-MSCs (KC+M-MSC group) or PBS vehicle (KC group) were directly injected into the bladder wall. One week after M-MSC injection, the therapeutic outcomes were evaluated via cystometry, histological analyses, and measurement of gene expression. Next, we compared the efficacy of M-MSCs at a low dose (1×10 cells) to that of an identical dose of adult bone marrow (BM)-derived MSCs.

Results: Rats in the KC group exhibited increased voiding frequency and reduced bladder capacity compared to rats of the sham group. However, these parameters recovered after transplantation of M-MSCs at all doses tested. KC bladders exhibited markedly increased mast cell infiltration, apoptosis, and tissue fibrosis. Administration of M-MSCs significantly reversed these characteristic histological alterations. Gene expression analyses indicated that several genes associated with tissue fibrosis were markedly upregulated in KC bladders. However the expression of these genes was significantly suppressed by the administration of M-MSCs. Importantly, M-MSCs ameliorated bladder deterioration in KC rats after injection of a low dose (1×10) of cells, at which point BM-derived MSCs did not substantially improve bladder function.

Conclusions: This study demonstrates for the first time the therapeutic efficacy of hESC-derived M-MSCs on KC in rats. M-MSCs restored bladder function more effectively than did BM-derived MSCs, protecting against abnormal changes including mast cell infiltration, apoptosis and fibrotic damage.
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http://dx.doi.org/10.5213/inj.1836014.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798637PMC
January 2018

Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix.

Biomaterials 2018 Jan 9;150:100-111. Epub 2017 Oct 9.

Center for Biomaterials, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea; Biomedical Engineering Major, Korea University of Science and Technology (UST), Daejeon, 34113, Republic of Korea. Electronic address:

Cell-derived matrices (CDM) are becoming an attractive alternative to conventional biological scaffolding platforms due to its unique ability to closely recapitulate a native extracellular matrix (ECM) de novo. Although cell-substrate interactions are recognized to be principal in regulating stem cell behavior, very few studies have documented the acclimation of human pluripotent stem cells (hPSCs) on pristine and altered cell-derived matrices. Here, we investigate crosslink-induced mechanotransduction of hPSCs cultivated on decellularized fibroblast-derived matrices (FDM) to explore cell adhesion, growth, migration, and pluripotency in various biological landscapes. The results showed either substrate-mediated induction or inhibition of the Epithelial-Mesenchymal-Transition (EMT) program, strongly suggesting that FDM stiffness can be a dominant factor in mediating hPSC plasticity. We further propose an optimal FDM substratum intended for long-term hPSC cultivation in a feeder-free niche-like microenvironment. This study carries significant implications for hPSC cultivation and encourages more in-depth studies towards the fundamentals of hPSC-CDM interactions.
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http://dx.doi.org/10.1016/j.biomaterials.2017.10.016DOI Listing
January 2018

Development of an alternative zebrafish model for drug-induced intestinal toxicity.

J Appl Toxicol 2018 Feb 13;38(2):259-273. Epub 2017 Oct 13.

Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, 08826, Republic of Korea.

An evaluation of intestinal toxicity is important because the mucosal lining of the gastrointestinal tract is the first barrier for oral xenobiotics. Until now, a rat model has been recommended as the standard intestinal toxicity model and the Caco-2 cell line, originated from a human colon adenocarcinoma, has been used as an alternative to this model, but there are limitations regarding cost-effectiveness and the need for mimicry of the human system. In this study, we investigated whether zebrafish could be a valid alternative to rats and Caco-2 cells as an intestinal toxicity model. We focused on intestinal gene expression of cytochrome P450 3A65, oxidative stress, apoptosis, inflammation, and intestinal function. Reverse transcription-quantitative polymerase chain reaction analysis was conducted using three models: zebrafish, Sprague-Dawley rats and Caco-2 cells, and the transcript levels and patterns of indicator genes were analyzed in conjunction with histopathological changes. Our results suggested that representative intestinal toxicants, indomethacin, diclofenac and methotrexate, induced significant transcript level changes in marker genes such as CYP3A, inducible nitric oxide synthase, heme oxygenase 1, superoxide dismutase 1, glutathione peroxidase 1, BCL2 associated X, B-cell lymphoma 2, caspase 9, tumor protein p53, nuclear factor-κB, interleukin-1β, tumor necrosis factor-alphaα and toll-like receptor 2 in the zebrafish model as in the rat and Caco-2 cells models. These results suggest that zebrafish model is sufficiently worth developing as an intestinal toxicity model that can replace or compensate the rat model or Caco-2 cell model.
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http://dx.doi.org/10.1002/jat.3520DOI Listing
February 2018