Publications by authors named "Kyoko Hidaka"

32 Publications

Cognitive Functions of Pediatric Brain Tumor Survivors Treated With Proton Beam Therapy: A Case Series.

J Pediatr Hematol Oncol 2020 Nov 23. Epub 2020 Nov 23.

Departments of Child Health.

Pediatric brain tumor survivors who received proton beam therapy at the University of Tsukuba Hospital from 2004 to 2011 were retrospectively evaluated for cognitive function. Five patients were included. The median age of diagnosis was 5.4 years (range: 1.5 to 12.5 y) and the median follow-up time was 5.8 years (range: 3.1 to 8.1 y). IQ scores at follow-up were decreased in 2 of 5 patients; 1 underwent whole-brain irradiation and the other was examined just after surgical removal of recurrent tumors. Local proton beam therapy may preserve cognitive function in survivors of pediatric brain tumors.
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http://dx.doi.org/10.1097/MPH.0000000000002011DOI Listing
November 2020

Corrigendum to "Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing" [Exp. Cell Res. 377 (2019) 24-35].

Exp Cell Res 2019 Oct 25;383(1):111432. Epub 2019 Jul 25.

Department of Medical Biophysics and Radiation Biology, Faculty of Medical Sciences, Kyushu University, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.yexcr.2019.05.013DOI Listing
October 2019

Geometrical Patterning and Constituent Cell Heterogeneity Facilitate Electrical Conduction Disturbances in a Human Induced Pluripotent Stem Cell-Based Platform: An Disease Model of Atrial Arrhythmias.

Front Physiol 2019 27;10:818. Epub 2019 Jun 27.

Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, Suita, Japan.

Ectopic foci from pulmonary veins (PVs) comprise the main trigger associated with the initiation of atrial fibrillation (AF). An abrupt anatomical narrow-to-wide transition, modeled as geometrical patterning with similar configuration in the present study, is located at the junction of PVs and the left atrium (LA). Complex cellular composition, i.e., constituent cell heterogeneity, is also observed in PVs and the PVs-LA junction. High frequency triggers accompanied with anatomical irregularity and constituent cell heterogeneity provoke impaired conduction, a prerequisite for AF genesis. However, few experiments investigating the effects of these factors on electrophysiological properties using human-based cardiomyocytes (CMs) with atrial properties have been reported. The aim of the current study was to estimate whether geometrical patterning and constituent cell heterogeneity under high frequency stimuli undergo conduction disturbance utilizing an two-dimensional (2D) monolayer preparation consisting of atrial-like CMs derived from human induced pluripotent stem cells (hiPSCs) and atrial fibroblasts (Fbs). We induced hiPSCs into atrial-like CMs using a directed cardiac differentiation protocol with the addition of all- retinoic acid (ATRA). The atrial-like hiPSC-derived CMs (hiPSC-CMs) and atrial Fbs were transferred in defined ratios (CMs/Fbs: 100%/0% or 70%/30%) on manually fabricated plates with or without geometrical patterning imitating the PVs-LA junction. High frequency field stimulation emulating repetitive ectopic foci originated in PVs were delivered, and the electrical propagation was assessed by optical mapping. We generated high purity CMs with or without the ATRA application. ATRA-treated hiPSC-CMs exhibited significantly higher atrial-specific properties by immunofluorescence staining, gene expression patterns, and optical action potential parameters than those of ATRA-untreated hiPSC-CMs. Electrical stimuli at a higher frequency preferentially induced impaired electrical conduction on atrial-like hiPSC-CMs monolayer preparations with an abrupt geometrical transition than on those with uniform geometry. Additionally, the application of human atrial Fbs to the geometrically patterned atrial-like hiPSC-CMs tended to further deteriorate the integrity of electrical conduction compared with those using the atrial-like hiPSC-CM alone preparations. Thus, geometrical narrow-to-wide patterning under high frequency stimuli preferentially jeopardized electrical conduction within atrial-like hiPSC-CM monolayers. Constituent cell heterogeneity represented by atrial Fbs also contributed to the further deterioration of conduction stability.
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http://dx.doi.org/10.3389/fphys.2019.00818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610482PMC
June 2019

Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.

Exp Cell Res 2019 04 22;377(1-2):24-35. Epub 2019 Feb 22.

Department of Medical Biophysics and Radiation Biology, Faculty of Medical Sciences, Kyushu University, Japan. Electronic address:

Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.
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http://dx.doi.org/10.1016/j.yexcr.2019.02.020DOI Listing
April 2019

Cited4 is related to cardiogenic induction and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis.

PLoS One 2017 17;12(8):e0183225. Epub 2017 Aug 17.

Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago, Japan.

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183225PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560578PMC
October 2017

Vestigial-like 2 contributes to normal muscle fiber type distribution in mice.

Sci Rep 2017 08 2;7(1):7168. Epub 2017 Aug 2.

Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka, 565-8565, Japan.

Skeletal muscle is composed of heterogeneous populations of myofibers that are classified as slow- and fast-twitch fibers. The muscle fiber-type is regulated in a coordinated fashion by multiple genes, including transcriptional factors and microRNAs (miRNAs). However, players involved in this regulation are not fully elucidated. One of the members of the Vestigial-like factors, Vgll2, is thought to play a pivotal role in TEA domain (TEAD) transcription factor-mediated muscle-specific gene expression because of its restricted expression in skeletal muscles of adult mice. Here, we generated Vgll2 null mice and investigated Vgll2 function in adult skeletal muscles. These mice presented an increased number of fast-twitch type IIb fibers and exhibited a down-regulation of slow type I myosin heavy chain (MyHC) gene, Myh7, which resulted in exercise intolerance. In accordance with the decrease in Myh7, down-regulation of miR-208b, encoded within Myh7 gene and up-regulation of targets of miR-208b, Sox6, Sp3, and Purβ, were observed in Vgll2 deficient mice. Moreover, we detected the physical interaction between Vgll2 and TEAD1/4 in neonatal skeletal muscles. These results suggest that Vgll2 may be both directly and indirectly involved in the programing of slow muscle fibers through the formation of the Vgll2-TEAD complex.
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http://dx.doi.org/10.1038/s41598-017-07149-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540913PMC
August 2017

Comorbidity and quality of life in childhood cancer survivors treated with proton beam therapy.

Pediatr Int 2017 Oct;59(10):1039-1045

Department of Child Health, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Background: The rate of childhood cancer survival has recently reached >80%. Various adverse events among childhood cancer survivors (CCS) have been reported. Proton beams are able to avoid unnecessary irradiation to normal/vital organs. We conducted a quality of life (QOL) study for CCS who were treated with proton beam therapy (PBT).

Methods: We included those patients treated with PBT to the brain, head, or neck and who were ≤15 years old at the University of Tsukuba Hospital between 1983 and 2011. Clinical information was collected from medical records. Questionnaires including the Pediatric Quality of Life Inventory (PedsQL) 4.0 Generic Core Scales (which assess health-related quality of life) were sent to the families/patients.

Results: Sixty patients were included. Median age at treatment was 6.2 years. The number of patients with status alive/dead/unknown was 32/24/4. Median follow-up period was 63.0 months (range, 48-340 months) for survivors. Questionnaires were sent to 25 families/patients and 19 were returned. PedsQL was assessed for 17 patients. Eleven of 32 living patients had at least one comorbidity grade 3/4. Average QOL score was above that for Japanese schoolchildren and adolescents. There was no correlation with comorbidity, and only longer time from treatment was correlated with a higher PedsQL score (P = 0.006).

Conclusion: CCS who were treated with multimodal treatment using PBT had a higher QOL score. Higher score was related to longer time since treatment, regardless of comorbidity.
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http://dx.doi.org/10.1111/ped.13323DOI Listing
October 2017

Electrophysiological properties of prion-positive cardiac progenitors derived from murine embryonic stem cells.

Circ J 2012 4;76(12):2875-83. Epub 2012 Sep 4.

Division of Regenerative Medicine and Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Yonago, Japan.

Background: The prion protein (PrP) has been reported to serve as a surface maker for isolation of cardiomyogenic progenitors from murine embryonic stem (ES) cells. Although PrP-positive cells exhibited automaticity, their electrophysiological characteristics remain unresolved. The aim of the present study was therefore to investigate the electrophysiological properties of PrP-positive cells in comparison with those of HCN4p-or Nkx2.5-positive cells.

Methods And Results: Differentiation of AB1, HCN5p-EGFP and hcgp7 ES cells into cardiac progenitors was induced by embryoid body (EB) formation. EBs were dissociated and cells expressing PrP, HCN4-EGFP and/or Nkx2.5-GFP were collected via flow cytometry. Sorted cells were subjected to reverse transcriptase-polymerase chain reaction, immunostaining and patch-clamp experiments. PrP-positive cells expressed mRNA of undifferentiation markers, first and second heart field markers, and cardiac-specific genes and ion channels, indicating their commitment to cardiomyogenic progenitors. PrP-positive cells with automaticity showed positive and negative chronotropic responses to isoproterenol and carbamylcholine, respectively. Hyperpolarization-activated cation current (I(f)) was barely detectable, whereas Na(+) and L-type Ca(2+) channel currents were frequently observed. Their spontaneous activity was slowed by inhibition of sarcoplasmic reticulum Ca(2+) uptake and release but not by blocking I(f). The maximum diastolic potential of their spontaneous firings was more depolarized than that of Nkx2.5-GFP-positive cells.

Conclusions: PrP-positive cells contained cardiac progenitors that separated from the lineage of sinoatrial node cells. PrP can be used as a marker to enrich nascent cardiac progenitors.
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http://dx.doi.org/10.1253/circj.cj-12-0126DOI Listing
May 2013

Different distribution of Cav3.2 and Cav3.1 transcripts encoding T-type Ca(2+) channels in the embryonic heart of mice.

Biomed Res 2010 Oct;31(5):301-5

Division of Molecular Medicine and Therapeutics, Department of Multidisciplinary Internal Medicine, Faculty of Medicine, Tottori University, Yonago, Japan.

We investigated the distribution of T-type Ca(2+) channel mRNAs in the mouse embryonic heart. Cav3.2, but not Cav3.1, was expressed in the E8.5 embryonic heart along with cardiac progenitor markers (Nkx2.5, Tbx5, Isl-1) and contractile proteins (alpha and beta MHC). In the E10.5 heart, the distribution of Cav3.1 mRNA was confirmed in the AV-canal and overlapped with that of MinK or Tbx2. Cav3.2 mRNA was observed not only in the AV-canal but also in the outflow tract, along with MinK and Isl-1, indicating the expression of Cav3.2 in the secondary heart field. Thus, Cav3.2 may contribute to the development of the outflow tract from the secondary heart field in the embryonic heart, whereas Cav3.1 may be involved in the development of the cardiac conduction-system together with Cav3.2.
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http://dx.doi.org/10.2220/biomedres.31.301DOI Listing
October 2010

RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation.

Genes Cells 2010 Nov;15(11):1158-67

Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.

The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12. RNA-binding proteins bind to specific target RNA sequences and regulate gene expression in a post-transcriptional manner. This study demonstrates that RNA-binding motif protein 24 (Rbm24) interacts with the 3'-untranslated region of myogenin mRNA and affects its half-life in C2C12 myogenesis. Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis. In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation. Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24-knockdown cells. Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells. Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin-dependent post-transcriptional regulatory pathway.
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http://dx.doi.org/10.1111/j.1365-2443.2010.01446.xDOI Listing
November 2010

Differentiation of pharyngeal endoderm from mouse embryonic stem cell.

Stem Cells Dev 2010 Nov 13;19(11):1735-43. Epub 2010 Sep 13.

Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.

Embryonic stem cells are considered to be a good in vitro tool to study the induction of various cell types including cardiomyocytes; however, induction of the pharyngeal endoderm (PE), the underlying heart-forming region, in vivo has been scarcely reported. In the present study, we found that many PE-related genes, such as Paxl, Pax9, Sixl, and Tbxl, were up-regulated in cardiomyocyte-rich embryoid bodies (EBs). The third pouch-related genes including Hoxa3, Foxn1, and Aire, which are crucial for thymus development and function, were also detected in later stages. Nkx2.5, a cardiac transcription factor gene, is known to be transiently expressed in the PE. By crossing Nkx2.5-Cre mice with Cre-dependent EGFP reporter mice, we found that Nkx2.5(+) lineage exclusively contributed to thymic epithelial cell development, followed by thymus development. Gene expression analysis using Nkx2.5-EGFP ES cells also revealed that PE-related mRNAs were specifically enriched in the transiently appearing E-cadherin(+)Nkx2.5(+) cell fraction. Interestingly, the EB-derived cells were found capable of supporting T-cell differentiation to CD4 or CD8 double-positive cells in a reaggregation organ culture in vitro. Our results suggest that EBs contain cells that resemble third pharyngeal pouch endoderm and confer a thymus-like microenvironment.
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http://dx.doi.org/10.1089/scd.2009.0466DOI Listing
November 2010

Nongenetic method for purifying stem cell-derived cardiomyocytes.

Nat Methods 2010 Jan 29;7(1):61-6. Epub 2009 Nov 29.

Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.
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http://dx.doi.org/10.1038/nmeth.1403DOI Listing
January 2010

The cellular prion protein identifies bipotential cardiomyogenic progenitors.

Circ Res 2010 Jan 12;106(1):111-9. Epub 2009 Nov 12.

Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.

Rationale: The paucity of specific surface markers for cardiomyocytes and their progenitors has impeded the development of embryonic or pluripotent stem cell-based transplantation therapy. Identification of relevant surface markers may also enhance our understanding of the mechanisms underlying differentiation.

Objective: Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors.

Methods And Results: Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential.

Conclusions: PrP+ cells from beating embryoid bodies (EBs) frequently included nascent Myhc+ cardiomyocytes. Cultured PrP+ cells further differentiated, giving rise to cardiac troponin I+ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR)alpha, enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP+PDGFRalpha+ (PRa) cells. The Myhc- PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc+, whereas they were Myhc- at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc- PRa cell-derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.109.209478DOI Listing
January 2010

RNA-binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21-dependent and -independent regulatory pathways.

Genes Cells 2009 Nov 8;14(11):1241-52. Epub 2009 Oct 8.

Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.

Skeletal muscle differentiation entails organized sequential events, including cell cycle arrest of proliferating myoblast cells and cell fusion, which lead to the formation of multinucleated myotubes. This process involves both transcriptional and post-transcriptional regulation of the gene expression of myogenic proteins, as well as cell-cycle related proteins. RNA-binding proteins bind to specific sequences of target RNA and regulate gene expression in a post-transcriptional manner. However, few tissue-specific RNA binding proteins have been identified. Herein, we report that the RNA binding proteins Rbm24 and Rbm38 were found to be preferentially expressed in muscle during differentiation in vitro. Further, knockdown of either by RNA interference suppressed cell-cycle arrest and delayed myogenic differentiation in C2C12 cells. In contrast, over-expression of Rbm24 or Rbm38 induced cell cycle arrest, and then had a positive effect on myogenic differentiation. Immunoprecipitation-RT-PCR analysis using tagged Rbm proteins indicated that Rbm38 binds to the p21 transcript in vivo. Consistent with this, differentiation of Rbm38 knockdown cells was rescued by over-expression of p21. Together, our results suggest that Rbm38 plays a crucial role in cell cycle arrest and myogenic differentiation via its binding to p21.
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http://dx.doi.org/10.1111/j.1365-2443.2009.01347.xDOI Listing
November 2009

Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells.

Biochem Biophys Res Commun 2008 Dec 18;377(2):413-418. Epub 2008 Oct 18.

Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.

For myocardial regeneration therapy, the low differentiation capability of functional cardiomyocytes sufficient to replace the damaged myocardial tissue is one of the major difficulties. Using Nkx2.5-GFP knock-in ES cells, we show a new efficient method to obtain cardiomyocytes from embryonic stem (ES) cells. The proportion of GFP-positive cells was significantly increased when ES cells were cultured with a conditioned medium from aortic endothelial cells (ECs), accompanied by upregulation of cardiac-specific genes as well as other mesodermal genes. The promotion was more prominent when EC-conditioned medium was added at an early stage of ES cell differentiation culture (Day 0-3). Inhibitors of bone morphogenic protein (BMP), cyclooxygenase (COX), and nitric oxide synthetase (NO) prevented the promotion of cardiomyogenesis by EC-conditioned medium. These results suggest that supplementation of EC-conditioned medium enables cardiomyocytes to be obtained efficiently through promotion of mesoderm induction, which is regulated by BMP, COX, and NOS.
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http://dx.doi.org/10.1016/j.bbrc.2008.09.160DOI Listing
December 2008

Changes of HCN gene expression and I(f) currents in Nkx2.5-positive cardiomyocytes derived from murine embryonic stem cells during differentiation.

Biomed Res 2008 Aug;29(4):195-203

Department of Cardiovascular Medicine, Tottori University Faculty of Medicine, Japan.

Changes in the expression of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels and I(f) currents during the differentiation of embryonic stem cells into cardiac cells remain unknown. We examined changes of HCN genes in expression and function during the differentiation of Nkx2.5-positive cardiac precursor cells derived from mouse ES cells using cell sorting, RTPCR, immunofluorescence and whole cell patch-clamp techniques. Cs(+)-induced inhibition of automaticity and transcription of HCN genes increased during differentiation. Expressions of Nkx2.5, a marker of cardiac progenitor cell, and Flk1, a marker of hemangioblast, were mutually exclusive. Messenger RNA and proteins encoded by HCN1 and 4 genes were predominantly observed in Nkx2.5-positive cells on day 15, although Flk1-positive cells did not express genes of the HCN family on that day. Cs(+)-induced prolongation of the cycle of spontaneous action potentials and I(f) currents were predominantly observed on day 15. These results suggested that a fraction of Nkx2.5-positive cardiac precursor cells was committed to pacemaking cells expressing I(f) channels predominantly encoded by HCN 1 and 4 genes.
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http://dx.doi.org/10.2220/biomedres.29.195DOI Listing
August 2008

Activation of MEK-ERK by heregulin-beta1 promotes the development of cardiomyocytes derived from ES cells.

Biochem Biophys Res Commun 2007 Sep 23;361(3):732-8. Epub 2007 Jul 23.

Department of Biology and Institute of Life Science and Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Republic of Korea. [corrected]

We have previously shown that heregulin-beta1 (HRG-beta1) was involved in the development and survival of cardiomyocytes derived from embryonic stem (ES) cells. This study was conducted to investigate the intracellular signal mechanisms by which HRG-beta1 stimulates cardiogenesis in ES cells. The treatment with ErbB receptor inhibitor decreased the population of cardiomyocytes and transcripts levels of cardiac genes (Nkx2.5, beta-MHC, cTnI, and MLC2a). The phosphorylation of ERK and development of cardiomyocytes by treatment with HRG-beta1 was suppressed upon treatment with MEK1 inhibitor. Furthermore, cardiomyocytes and level of MHC protein were significantly increased by overexpression of wild type MEK1 or constitutive active MEK1, but not dominant negative MEK1. These results suggest that HRG-beta1 promotes the development of cardiomyocytes predominantly by activation of MEK-ERK.
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http://dx.doi.org/10.1016/j.bbrc.2007.07.045DOI Listing
September 2007

Endocardiogenesis in embryoid bodies: novel markers identified by gene expression profiling.

Biochem Biophys Res Commun 2007 Jun 17;357(4):896-902. Epub 2007 Apr 17.

Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.

Endocardial cells and cardiomyocytes differentiate from the cardiogenic mesoderm at about the same time during development. Although in vitro embryonic stem (ES) cell systems have been used to study the differentiation of various types of cell lineages, including cardiomyocytes, smooth muscle cells, and vascular endothelial cells, differentiation of endocardial cells, or endocardiogenesis, has not been well reported, because of a lack of specific molecular markers. In our search for cardiogenesis-associated genes expressed in embryoid bodies, we found several genes expressed in the heart region of mouse embryos, but not in cardiomyocytes. To identify the cell types expressing these genes, CD31(+) cells were taken from mouse embryos on embryonic day (E)8.5 and E9.5 and sorted, then their transcripts were analyzed using quantitative RT-PCR analyses. In those embryos, Gata4 and Nfatc1, as well as newly identified Cgnl1 and Dok4 were found to be preferentially expressed in endocardial cells, but not in yolk sac endothelial cells, while Cdh5 and Kdr were expressed in both cardiac and yolk sac endothelial cells. Immunohistochemical analyses of embryoid bodies revealed that some CD31(+) cells co-expressing Gata4 and Nfatc1 were located in close proximity to cardiomyocytes. These results suggest that embryoid bodies express endocardial specific genes and likely generate endocardial cells along with cardiomyocytes. Further, they indicate that these new marker genes are useful to study the origin and induction of endocardial cells, and identify other endocardial markers.
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http://dx.doi.org/10.1016/j.bbrc.2007.04.030DOI Listing
June 2007

Efficient capture of cardiogenesis-associated genes expressed in ES cells.

Biochem Biophys Res Commun 2007 Mar 30;355(1):47-53. Epub 2007 Jan 30.

Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.

Cardiogenesis can be induced in vitro in ES cells, though it is difficult to distinguish cardiac-specific genes, since embryoid bodies simultaneously differentiate into multiple lineages. In the present study, transient serum removal during culture greatly enhanced cardiogenesis, and reduced generation of endothelial and hematopoietic cells. Using DNA microarray analysis of 24 differentiated sample cultures including cardiogenesis-enhanced cells, we successfully selected genes up-regulated in embryoid bodies that had undergone cardiogenic differentiation. Besides contractile protein genes, cardiac transcriptional regulatory genes, such as Nkx2-5, Gata4/5, Mef2c, and Myocd, were primary constituents of the first 100 genes chosen as cardiogenesis-associated genes. Further, whole mount in situ hybridization analysis of 13 genes containing non-characterized ones confirmed that most of them were specifically expressed in the heart region of mouse embryos from E9.5-10.5. Based on our results, we consider that the present profiling method may be useful to identify novel genes important for cardiac development.
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http://dx.doi.org/10.1016/j.bbrc.2007.01.109DOI Listing
March 2007

Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis.

Proc Natl Acad Sci U S A 2006 Dec 14;103(52):19812-7. Epub 2006 Dec 14.

Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

Although Wingless (Wg)/Wnt signaling has been implicated in heart development of multiple organisms, conflicting results have been reported regarding the role of Wnt/beta-catenin pathway in cardiac myogenesis: Wg/armadillo signaling promotes heart development in Drosophila, whereas activation of Wnt/beta-catenin signaling inhibits heart formation in avians and amphibians. Using an in vitro system of mouse ES cell differentiation into cardiomyocytes, we show here that Wnt/beta-catenin signaling exhibits developmental stage-specific, biphasic, and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis. Activation of the Wnt/beta-catenin pathway in the early phase during embryoid body (EB) formation enhances ES cell differentiation into cardiomyocytes while suppressing the differentiation into hematopoietic and vascular cell lineages. In contrast, activation of Wnt/beta-catenin signaling in the late phase after EB formation inhibits cardiomyocyte differentiation and enhances the expression of hematopoietic/vascular marker genes through suppression of bone morphogenetic protein signaling. Thus, Wnt/beta-catenin signaling exhibits biphasic and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis, depending on the stage of development.
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http://dx.doi.org/10.1073/pnas.0605768103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1750922PMC
December 2006

Delayed onset of beating and decreased expression of T-type Ca2+ channel in mouse ES cell-derived cardiocytes carrying human chromosome 21.

Biochem Biophys Res Commun 2006 Dec 12;351(1):126-32. Epub 2006 Oct 12.

Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Tottori University Graduate School of Medical Science, 86 Nishi-cho, Yonago 683-8503, Japan.

The mouse ES cell line hcgp7/#21, which carries a human chromosome 21 (hChr.21), was used as an in vitro model to examine the effects of hChr.21 on cardiomyocyte differentiation. Cardiomyocytes derived from hcgp7/#21 showed a significant delay in the onset of spontaneous beating. The number of Nkx2.5/GFP(+) cardiac progenitor cells was comparable to that in control ES cells and they also expressed comparable mRNA levels for mesodermal markers, cardiac specific transcription factors, contractile proteins, and L-type Ca(2+) channels. However, cells from hcgp7/#21 expressed significantly reduced levels of mRNA for Cav3.1 and Cav3.2, which was consistent with the decreased number of cells expressing T-type Ca(2+) channels and decreased T-type Ca(2+) channel currents. These findings suggest that the presence of human chromosome 21 suppresses expression of T-type Ca(2+) channels in cardiomyocytes during differentiation, which may be responsible for delayed onset of spontaneous beating.
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http://dx.doi.org/10.1016/j.bbrc.2006.10.018DOI Listing
December 2006

[Challenges towards regenerative medicine].

Nihon Rinsho 2006 May;64(5):990-5

Department of Bioscience, National Cardiovascular Center Research Institute.

Regenerative medicine is a promising approach to treat patients with severe cardiac failure. Since embryonic stem cells (ES cells) easily differentiate into cardiomyocytes, ES cells are thought to be a good candidate resource for cardiac cell transplantation therapy. However, molecular mechanism of cardiac differentiation is still largely unknown. Here we discuss our present approach to understand the mechanism of cardiogenesis at the molecular level as well as novel genes and cascades that are important for cardiac differentiation. Further observation will help to establish the new strategy of regenerative medicine for patients with cardiac failure.
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May 2006

Cardiac ischemia activates vascular endothelial cadherin promoter in both preexisting vascular cells and bone marrow cells involved in neovascularization.

Circ Res 2006 Apr 16;98(7):897-904. Epub 2006 Mar 16.

Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka, Japan.

Vascular endothelial cadherin (VE-cadherin) is expressed on vascular endothelial cells, which are involved in developmental vessel formation. However, it remains elusive how VE-cadherin-expressing cells function in postnatal neovascularization. To trace VE-cadherin-expressing cells, we developed mice expressing either green fluorescent protein or LacZ driven by VE-cadherin promoter using Cre-loxP system. Although VE-cadherin promoter is less active after birth than during embryogenesis in blood vessels, it is reactivated on cardiac ischemia. Both types of reporter-positive cells are found in the vasculature and in the infarcted myocardium. Those found in the vasculature were pre-existing endothelial cells and incorporated endothelial progenitor cells derived from extracardiac tissue. In addition to the vasculature, VE-cadherin promoter-activated cells were positive for CD45 in the bone marrow cells of the infarcted mice. VE-cadherin promoter-reactivated CD45-positive leukocytes were also found in the infarcted area. In addition, VE-cadherin promoter was activated in the bone marrow vessels of the infarcted mice. Collectively, our findings reveal a new ischemia-induced neovascularization mechanism involving VE-cadherin; the re-expressed VE-cadherin-mediated cell adhesion between cells may be involved not only in homing of bone marrow-derived cells to ischemic area but also mobilization from bone marrow.
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http://dx.doi.org/10.1161/01.RES.0000218193.51136.adDOI Listing
April 2006

Impairment of cardiomyogenesis in embryonic stem cells lacking scaffold protein JSAP1.

Biochem Biophys Res Commun 2005 Dec 21;338(2):1152-7. Epub 2005 Oct 21.

Division of Cell Cycle Regulation, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, is important in embryonic stem (ES) cells during neurogenesis. In that study, we also observed the altered expression of mesodermal marker genes, which indicated that JSAP1 is involved in the differentiation of mesodermal lineages. Here, we investigated the function of JSAP1 in cardiomyocyte development using JSAP1-null ES cells, and found that cardiomyogenesis was impaired in the JSAP1-null mutant. The JSAP1 deficiency resulted in lower gene expression of the cardiac transcription factor Nkx2.5 and contractile proteins. In contrast, the mutant showed a significantly higher expression of mesoderm-related markers other than those of the cardiomyocyte lineage. Together, these results suggest that JSAP1 may be important for the differentiation of the mesodermal lineages, functioning as a positive factor for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into other lineages.
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http://dx.doi.org/10.1016/j.bbrc.2005.10.052DOI Listing
December 2005

Subtype switching of T-type Ca 2+ channels from Cav3.2 to Cav3.1 during differentiation of embryonic stem cells to cardiac cell lineage.

Circ J 2005 Oct;69(10):1284-9

Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Japan.

Background: The developmental changes of Ni(2+)-sensitivity to automaticity of Nkx2.5-positive cells derived from mouse embryonic stem cell have been identified, suggesting developmental regulation of expressing Ni(2+)-sensitive T-type Ca(2+) channel, although the mechanism of the change has not been fully studied.

Methods And Results: Transcripts of Cav3.2, Cav3.1 and Cav1.2 genes of beating Nkx2.5-positive cells, which encode the Ni(2+)-sensitive T-type Ca(2+) channel, Ni(2+)-insensitive T-type Ca(2+) channel, and L-type Ca(2+) channel, respectively, were investigated by real-time reverse-transcriptase-polymerase chain reaction, and the current density of each channel was measured by patch-clamp techniques at the early and late stages of differentiation. The expression of the Cav3.2 transcript predominated in the early stage whereas those of Cav3.1 and Cav1.2 transcripts were upregulated in the late stage, which was consistent with the change in each current density, suggesting the expression of channel proteins is largely determined at the transcriptional level.

Conclusion: The results indicate that the mechanism of change of Ni(2+)-sensitivity is partly, if not completely, the subtype switch of T-type Ca(2+) channel from Cav3.2 to Cav3.1 at the transcriptional level, and that the expression of the L-type Ca(2+) channel might have an attenuating effect on Ni(2+)-sensitivity to automaticity in the late stage of differentiation.
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http://dx.doi.org/10.1253/circj.69.1284DOI Listing
October 2005

Stable and uniform gene suppression by site-specific integration of siRNA expression cassette in murine embryonic stem cells.

Stem Cells 2005 Sep 7;23(8):1028-34. Epub 2005 Jun 7.

Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.

We developed a simple system to introduce small interfering RNA (siRNA) into murine embryonic stem cells (ESCs) and then showed its stable and uniform expression. Using hypoxanthine guanine phosphoribosyl transferase 1 (Hprt)-deficient ESCs as a recipient, we efficiently introduced an siRNA expression cassette into the Hprt locus by homologous recombination, which was easily detected by HAT selection. Nearly all of the HAT-resistant clones exhibited a silenced expression of the exogenous target gene (enhanced green fluorescent protein [EGFP]) or the endogenous target gene (Flk1). Flow cytometry profiles demonstrated that there were no significant differences in level of suppression among individual clones and cells. The suppressing effect by siRNA was maintained for more than 1 month in both undifferentiated and differentiated ESCs, while its persistent expression did not disturb their growth or differentiation potential. The stable and uniform suppression capability of this system will help to screen genes and provide important information regarding cell differentiation in ESCs.
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http://dx.doi.org/10.1634/stemcells.2004-0335DOI Listing
September 2005

Acetylation of GATA-4 is involved in the differentiation of embryonic stem cells into cardiac myocytes.

J Biol Chem 2005 May 13;280(20):19682-8. Epub 2005 Mar 13.

Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Japan.

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for acetylation and DNA binding of GATA-4 and its full transcriptional activity as well as for promotion of a transcriptionally active chromatin configuration. However, the roles of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of ES cells into cardiac myocytes remain unknown. In an ES cell model of developing embryoid bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. Finally, TSA facilitated the expression of green fluorescence protein under the control of the cardiac-specific Nkx-2.5 promoter and of endogenous cardiac beta-myosin heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histones is involved in the differentiation of ES cells into cardiac myocytes.
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http://dx.doi.org/10.1074/jbc.M412428200DOI Listing
May 2005

Wnt11 facilitates embryonic stem cell differentiation to Nkx2.5-positive cardiomyocytes.

Biochem Biophys Res Commun 2004 Dec;325(3):968-75

Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.

Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.
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http://dx.doi.org/10.1016/j.bbrc.2004.10.103DOI Listing
December 2004

[Induction and isolation of embryonic stem cell-derived cradiomyocytes].

Authors:
Kyoko Hidaka

Fukuoka Igaku Zasshi 2004 Jul;95(7):153-9

Department of Bioscience, National Cardiovascular Center, Research Institute.

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July 2004

Developmental changes of Ni(2+) sensitivity and automaticity in Nkx2.5-positive cardiac precursor cells from murine embryonic stem cell.

Circ J 2004 Jul;68(7):724-6

Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Tottori, Japan.

Background: It is controversial which subtypes of T type Ca(2+) channels are implicated in automaticity of cardiac cells during the embryonic period.

Method And Results: The effect of Ni(2+) on the automaticity of Nkx2.5-positive cardiac precursor cells sorted from embryonic stem cells during their differentiation was examined using patch clamp techniques. Although 40 micromol/L Ni(2+), which is enough to block Ni(2+)sensitive T type-Ca(2+) channels, decreased the spontaneous beating rate in all cells in the early and intermediate stage, Ni(2+) did not show any effects on the automaticity of 50% of the cells in the late stage.

Conclusion: These results indicate that Ni(2+)-sensitive T-type Ca(2+) channels expressed in the Nkx2.5-positive cardiac precursor cells are developmentally regulated.
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http://dx.doi.org/10.1253/circj.68.724DOI Listing
July 2004