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1.
Biochem Biophys Res Commun ; 574: 91-96, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34450429

RESUMO

A major concern in the clinical application of induced pluripotent stem cells (iPSCs) is the prevention of tumorigenesis after implantation. Stem cells with high proliferative and differentiation potential are sensitive to radiation. Therefore, we hypothesized that irradiation may selectively eliminate residual undifferentiated human iPSCs (hiPSCs) in a cell population containing differentiated cardiomyocytes derived from hiPSCs (hiPSCs-CMs) and thus reduce tumorigenicity in vivo. hiPSC-CMs were irradiated with X-rays, after which the cell proliferation, apoptosis, morphology, and gene expression were analyzed. The gene expression of Lin28A, Nanog, Oct3/4, and SRY-box 2 was significantly lower in the irradiation group than in the control group. Irradiated hiPSC-CMs showed no change in proliferation potency and morphology compared to untreated hiPSC-CMs. Furthermore, irradiation did not induce apoptosis of differentiated cardiomyocytes. No significant difference in the gene expression of cardiac-specific markers, including α-myosin heavy chain, cardiac troponin T, and NK2 Homeobox 5, was observed between the groups. Tumorigenicity tests using NOG mice showed less frequent tumor formation in the irradiation group than in the control group. Irradiation of hiPSC-CMs significantly reduced the number of undifferentiated hiPSC and the tumor formation, while minimizing any adverse effects on hiPSC-CMs, thereby enabling safe hiPSC-based treatment.

2.
Methods Mol Biol ; 2320: 29-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302645

RESUMO

The human adult heart consists of approximately four billion cardiomyocytes, which do not possess self-renewal abilities. Severe myocardial infarction and dilated cardiomyopathy result in the loss of more than a billion cardiomyocytes. Induced pluripotent stem cells (iPSCs) can differentiate into various types of cells. Due to this ability, these cells could potentially serve as a new resource for cell therapy. Many studies have utilized cardiomyocytes derived from iPSCs for myocardial regeneration therapy. To obtain large number of cardiomyocytes for transplantation, we need to develop effective methods that would allow us to dissociate multiple cardiomyocyte aggregates simultaneously. Here, we describe a method to efficiently dissociate large number of iPSC-derived cardiomyocyte aggregates.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Infarto do Miocárdio/terapia
3.
J Am Heart Assoc ; 10(13): e008649, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34212772

RESUMO

Background Clinical effectiveness of autologous skeletal cell-patch implantation for nonischemic dilated cardiomyopathy has not been clearly elucidated in clinical settings. This clinical study aimed to determine the feasibility, safety, therapeutic efficacy, and the predictor of responders of this treatment in patients with nonischemic dilated cardiomyopathy. Methods and Results Twenty-four nonischemic dilated cardiomyopathy patients with left ventricular ejection fraction <35% on optimal medical therapy were enrolled. Autologous cell patches were implanted over the surface of the left ventricle through left minithoracotomy without procedure-related complications and lethal arrhythmia. We identified 13 responders and 11 nonresponders using the combined indicator of a major cardiac adverse event and incidence of heart failure event. In the responders, symptoms, exercise capacity, and cardiac performance were improved postoperatively (New York Heart Association class II 7 [54%] and III 6 [46%] to New York Heart Association class II 12 [92%] and I 1 [8%], P<0.05, 6-minute walk test; 471 m [370-541 m] to 525 m [425-555 m], P<0.05, left ventricular stroke work index; 31.1 g·m2·beat [22.7-35.5 g·m2·beat] to 32.8 g·m2·beat [28-38.5 g·m2·beat], P=0.21). However, such improvement was not observed in the nonresponders. In responders, the actuarial survival rate was 90.9±8.7% at 5 years, which was superior to the estimated survival rate of 70.9±5.4% using the Seattle Heart Failure Model. However, they were similar in nonresponders (47.7±21.6% and 56.3±8.1%, respectively). Multivariate regression model with B-type natriuretic peptide, pulmonary capillary wedge pressure, and expression of histone H3K4me3 (H3 lysine 4 trimethylation) strongly predicted the responder of this treatment (B-type natriuretic peptide: odds ratio [OR], 0.96; pulmonary capillary wedge pressure: ​OR, 0.58; H3K4me3: OR, 1.35, receiver operating characteristic-area under the curve, 0.96, P<0.001). Conclusions This clinical trial demonstrated that autologous skeletal stem cell-patch implantation might promise functional recovery and good clinical outcome in selected patients with nonischemic dilated cardiomyopathy, in addition to safety and feasibility. Registration URL: http://www.umin.ac.jp/english/. Unique identifiers: UMIN000003273, UMIN0000012906 and UMIN000015892.

4.
Hum Mol Genet ; 30(15): 1384-1397, 2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-33949662

RESUMO

Desmoglein-2, encoded by DSG2, is one of the desmosome proteins that maintain the structural integrity of tissues, including heart. Genetic mutations in DSG2 cause arrhythmogenic cardiomyopathy, mainly in an autosomal dominant manner. Here, we identified a homozygous stop-gain mutations in DSG2 (c.C355T, p.R119X) that led to complete desmoglein-2 deficiency in a patient with severe biventricular heart failure. Histological analysis revealed abnormal deposition of desmosome proteins, disrupted intercalated disk structures in the myocardium. Induced pluripotent stem cells (iPSCs) were generated from the patient (R119X-iPSC), and the mutated DSG2 gene locus was heterozygously corrected to a normal allele via homology-directed repair (HDR-iPSC). Both isogenic iPSCs were differentiated into cardiomyocytes [induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)]. Multielectrode array analysis detected abnormal excitation in R119X-iPSC-CMs but not in HDR-iPSC-CMs. Micro-force testing of three-dimensional self-organized tissue rings (SOTRs) revealed tissue fragility and a weak maximum force in SOTRs from R119X-iPSC-CMs. Notably, these phenotypes were significantly recovered in HDR-iPSC-CMs. Myocardial fiber structures in R119X-iPSC-CMs were severely aberrant, and electron microscopic analysis confirmed that desmosomes were disrupted in these cells. Unexpectedly, the absence of desmoglein-2 in R119X-iPSC-CMs led to decreased expression of desmocollin-2 but no other desmosome proteins. Adeno-associated virus-mediated replacement of DSG2 significantly recovered the contraction force in SOTRs generated from R119X-iPSC-CMs. Our findings confirm the presence of a desmoglein-2-deficient cardiomyopathy among clinically diagnosed dilated cardiomyopathies. Recapitulation and correction of the disease phenotype using iPSC-CMs provide evidence to support the development of precision medicine and the proof of concept for gene replacement therapy for this cardiomyopathy.

5.
Sci Rep ; 11(1): 5654, 2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707655

RESUMO

We hypothesized that an appropriate ratio of cardiomyocytes, fibroblasts, endothelial cells, and extracellular matrix (ECM) factors would be required for the development of three-dimensional cardiac tissues (3D-CTs) as drug screening systems. To verify this hypothesis, ECM-coated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), ECM-coated cardiac fibroblasts (CFs), and uncoated cardiac endothelial cells (CEs) were mixed in the following ratios: 10:0:0 (10CT), 7:2:1 (7CT), 5:4:1 (5CT), and 2:7:1 (2CT). The expression of cardiac-, fibroblasts-, and endothelial-specific markers was assessed by FACS, qPCR, and immunostaining while that of ECM-, cell adhesion-, and ion channel-related genes was examined by qPCR. Finally, the contractile properties of the tissues were evaluated in the absence or presence of E-4031 and isoproterenol. The expression of ECM- and adhesion-related genes significantly increased, while that of ion channel-related genes significantly decreased with the CF proportion. Notably, 7CT showed the greatest contractility of all 3D-CTs. When exposed to E-4031 (hERG K channel blocker), 7CT and 5CT showed significantly decreased contractility and increased QT prolongation. Moreover, 10CT and 7CT exhibited a stronger response to isoproterenol than did the other 3D-CTs. Finally, 7CT showed the highest drug sensitivity among all 3D-CTs. In conclusion, 3D-CTs with an appropriate amount of fibroblasts/endothelial cells (7CT in this study) are suitable drug screening systems, e.g. for the detection of drug-induced arrhythmia.

6.
Sci Rep ; 9(1): 4638, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874579

RESUMO

Selection of human induced pluripotent stem cell (hiPSC) lines with high cardiac differentiation potential is important for regenerative therapy and drug screening. We aimed to identify biomarkers for predicting cardiac differentiation potential of hiPSC lines by comparing the gene expression profiles of six undifferentiated hiPSC lines with different cardiac differentiation capabilities. We used three platforms of gene expression analysis, namely, cap analysis of gene expression (CAGE), mRNA array, and microRNA array to efficiently screen biomarkers related to cardiac differentiation of hiPSCs. Statistical analysis revealed candidate biomarker genes with significant correlation between the gene expression levels in the undifferentiated hiPSCs and their cardiac differentiation potential. Of the candidate genes, PF4 was validated as a biomarker expressed in undifferentiated hiPSCs with high potential for cardiac differentiation in 13 additional hiPSC lines. Our observations suggest that PF4 may be a useful biomarker for selecting hiPSC lines appropriate for the generation of cardiomyocytes.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Fator Plaquetário 4/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , Organogênese/fisiologia , Fator Plaquetário 4/genética , RNA Mensageiro/genética , Transcriptoma/genética
7.
Sci Rep ; 9(1): 1881, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760836

RESUMO

Transplantation of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC-CMs) is a promising treatment for heart failure, but residual undifferentiated hiPSCs and malignant transformed cells may lead to tumor formation. Here we describe a highly sensitive tumorigenicity assay for the detection of these cells in hiPSC-CMs. The soft agar colony formation assay and cell growth analysis were unable to detect malignantly transformed cells in hiPSC-CMs. There were no karyotypic abnormalities during hiPSCs subculture and differentiation. The hiPSC markers TRA1-60 and LIN28 showed the highest sensitivity for detecting undifferentiated hiPSCs among primary cardiomyocytes. Transplantation of hiPSC-CMs with a LIN28-positive fraction > 0.33% resulted in tumor formation in nude rats, whereas no tumors were formed when the fraction was < 0.1%. These findings suggested that combination of these in vitro and in vivo tumorigenecity assays can verify the safety of hiPSC-CMs for cell transplantation therapy.


Assuntos
Testes de Carcinogenicidade/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Glicoproteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Neoplasias/etiologia , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Nus , Transplante/efeitos adversos
8.
Stem Cells Int ; 2019: 1271682, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31949433

RESUMO

Regenerative medicine has continued to progress for lung biology and lung diseases. Efforts have focused on a variety of different applications for pluripotent stem cells. Several groups have reported successful methods for inducing differentiation of induced pluripotent stem cells (iPSCs) into the airway epithelium such as alveolar epithelium type II (ATII). However, differentiation efficiency varies among reports and improvements are needed. In the present paper, we propose a novel method for elimination of residual undifferentiated murine iPSCs using JQ1, a potent inhibitor of bromodomain (BRD) and extraterminal domain (BET) family proteins, for efficient differentiation into ATII. First, the murine iPSC line 20D-17 was induced to differentiate into ATII over a period of 26 days (days 0-26) using previously reported embryoid body seeding and stepwise differentiation methods. mRNA expressions of differentiation markers including surfactant protein C (Sftpc) were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) results, and 17% of the cells were shown positive for prosurfactant protein C (proSPC) in flow cytometry analysis. Next, those cells were cultured three-dimensionally in Matrigel for an additional 14 days (days 26-40), during which JQ1 was added for 4 days (days 28-32) to remove residual undifferentiated iPSCs. As a result, on day 40, the mRNA expression level of Sftpc in the three-dimensional culture was maintained at the same level as on day 26 and shown to be further increased by the addition of JQ1, with 39% of the cells found to express proSPC, showing that differentiation efficiency could be further increased. Three-dimensional culture with BRD4 inhibition by JQ1 improved the differentiation induction efficiency to ATII by removing residual undifferentiated murine iPSCs during the differentiation induction process.

9.
Transplantation ; 103(2): 291-298, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30119058

RESUMO

BACKGROUND: Somatic stem cell (SC) therapy can improve cardiac performance following ischemic injury. In this study, we investigated whether induced pluripotent SC-derived cardiomyocytes (iPS-CMs) are more effective than somatic SCs, such as skeletal myoblasts (SM) and mesenchymal (M)SCs, in promoting functional recovery upon transplantation in a porcine model of myocardial infarction. METHODS: Myocardial injury was induced by ameroid ring placement in immunosuppressed female mini pigs; after 1 month, epicardial cell transplantation was performed with iPS-CMs (n = 7), SMs (n = 7), and MSCs (n = 7). Control pigs underwent sham operation (n = 8). RESULTS: Cell therapy improved functional recovery 2 months after myocardial infarction, as evidenced by increased ejection fraction (iPS-CM, +7.3% ± 2.2% and SM, +5.8% ± 5.4% vs control, -4.4% ± 3.8%; P < 0.05). The analysis of regional contractile function in the infarcted zone revealed an increase in transverse peak strain (iPS-CM, +4.6% ± 2.2% vs control, -3.8% ± 4.7%; P < 0.05). The C-11 acetate kinetic analysis by positron emission tomography showed that the work-metabolic cardiac energy efficacy increased by the transplantation of iPS-CMs, but was reduced by the other cell types. This was accompanied by decreased myocardial wall stress in the infarcted zone (iPS-CM, -27.6 ± 32.3 Pa and SM, -12.8 ± 27 Pa vs control, +40.5 ± 33.9 Pa; P < 0.05). CONCLUSIONS: The iPS-CM is superior to other somatic cell sources in terms of improving regional contractile function and cardiac bioenergetic efficiency, suggesting greater clinical benefits in severely damaged myocardium.


Assuntos
Coração/fisiopatologia , Células-Tronco Pluripotentes Induzidas/citologia , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Miócitos Cardíacos/transplante , Consumo de Oxigênio , Transplante de Células-Tronco/métodos , Animais , Apoptose , Modelos Animais de Doenças , Humanos , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Suínos
10.
mSystems ; 3(5)2018.
Artigo em Inglês | MEDLINE | ID: mdl-30417107

RESUMO

Bacillus anthracis is a Gram-positive endospore-forming bacterial species that causes anthrax in both humans and animals. In Zambia, anthrax cases are frequently reported in both livestock and wildlife, with occasional transmission to humans, causing serious public health problems in the country. To understand the genetic diversity of B. anthracis strains in Zambia, we sequenced and compared the genomic DNA of B. anthracis strains isolated across the country. Single nucleotide polymorphisms clustered these strains into three groups. Genome sequence comparisons revealed a large deletion in strains belonging to one of the groups, possibly due to unequal crossing over between a pair of rRNA operons. The deleted genomic region included genes conferring resistance to bacitracin, and the strains with the deletion were confirmed with loss of bacitracin resistance. Similar deletions between rRNA operons were also observed in a few B. anthracis strains phylogenetically distant from Zambian strains. The structure of bacitracin resistance genes flanked by rRNA operons was conserved only in members of the Bacillus cereus group. The diversity and genomic characteristics of B. anthracis strains determined in this study would help in the development of genetic markers and treatment of anthrax in Zambia. IMPORTANCE Anthrax is caused by Bacillus anthracis, an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.

11.
Sci Rep ; 8(1): 3726, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487310

RESUMO

Induced pluripotent stem cells (iPSCs) are promising candidate cells for cardiomyogenesis in the failing heart. However, teratoma/tumour formation originating from undifferentiated iPSCs contaminating the graft is a critical concern for clinical application. Here, we hypothesized that brentuximab vedotin, which targets CD30, induces apoptosis in tumourigenic cells, thus increasing the safety of iPSC therapy for heart failure. Flow cytometry analysis identified consistent expression of CD30 in undifferentiated human iPSCs. Addition of brentuximab vedotin in vitro for 72 h efficiently induced cell death in human iPSCs, associated with a significant increase in G2/M phase cells. Brentuximab vedotin significantly reduced Lin28 expression in cardiomyogenically differentiated human iPSCs. Transplantation of human iPSC-derived cardiomyocytes (CMs) without treatment into NOG mice consistently induced teratoma/tumour formation, with a substantial number of Ki-67-positive cells in the graft at 4 months post-transplant, whereas iPSC-derived CMs treated with brentuximab vedotin prior to the transplantation did not show teratoma/tumour formation, which was associated with absence of Ki-67-positive cells in the graft over the same period. These findings suggest that in vitro treatment with brentuximab vedotin, targeting the CD30-positive iPSC fraction, reduced tumourigenicity in human iPSC-derived CMs, potentially providing enhanced safety for iPSC-based cardiomyogenesis therapy in clinical scenarios.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígeno Ki-1/antagonistas & inibidores , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco , Animais , Apoptose/efeitos dos fármacos , Brentuximab Vedotin , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/imunologia , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Imunoconjugados/farmacologia , Antígeno Ki-1/genética , Antígeno Ki-1/metabolismo , Camundongos , Transplante de Células-Tronco/métodos
12.
Tissue Eng Part A ; 24(3-4): 287-300, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28498040

RESUMO

Although engineered cardiac tissues (ECTs) derived from induced pluripotent stem cells (iPSCs) are promising for myocardial regenerative therapy, the appropriate ratio of cardiomyocytes to non-cardiomyocytes is not fully understood. Here, we determined whether ECT-cell content is a key determinant of its structure/function, thereby affecting ECT therapeutic potential for advanced heart failure. Scaffold-free ECTs containing different ratios (25%, 50%, 70%, or 90%) of iPSC-derived cardiomyocytes were generated by magnetic-activated cell sorting by using cardiac-specific markers. Notably, ECTs showed synchronized spontaneous beating when cardiomyocytes constituted ≥50% of total cells, with the electrical-conduction velocity increasing depending on cardiomyocyte ratio; however, ECTs containing 90% cardiomyocytes failed to form stable structures. ECTs containing 25% or 50% cardiomyocytes predominantly expressed collagen and fibronectin, whereas ECTs containing 70% cardiomyocytes predominantly expressed laminin and exhibited the highest contractile/relaxation properties. Furthermore, transplantation of ECTs containing 50% or 70% cardiomyocytes into a rat chronic myocardial infarction model led to a more profound functional recovery as compared with controls. Notably, transplanted ECTs showed electrical synchronization with the native heart under Langendorff perfusion. Collectively, these results indicate that the quantity of non-cardiomyocytes is critical in generating functional iPSC-derived ECTs as grafts for cardiac-regeneration therapy, with ECTs containing 50-70% cardiomyocytes exhibiting stable structures and increased cardiotherapeutic potential.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Citometria de Fluxo , Humanos , Engenharia Tecidual/métodos
13.
Tissue Eng Part C Methods ; 24(1): 56-67, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28967302

RESUMO

An in vitro drug-induced cardiotoxicity assay is a critical step in drug discovery for clinical use. The use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is promising for this purpose. However, single hiPSC-CMs are limited in their ability to mimic native cardiac tissue structurally and functionally, and the generation of artificial cardiac tissue using hiPSC-CMs is an ongoing challenging. We therefore developed a new method of constructing three-dimensional (3D) artificial tissues in a short time by coating extracellular matrix (ECM) components on cell surfaces. We hypothesized that 3D cardiac tissues derived from hiPSC-CMs (3D-hiPSC-CT) could be used for an in vitro drug-induced cardiotoxicity assay. 3D-hiPSC-CT were generated by fibronectin and gelatin nanofilm coated single hiPSC-CMs. Histologically, 3D-hiPSC-CT exhibited a sarcomere structure in the myocytes and ECM proteins, such as fibronectin, collagen type I/III, and laminin. The administration of cytotoxic doxorubicin at 5.0 µM induced the release of lactate dehydrogenase, while that at 2.0 µM reduced the cell viability. E-4031, human ether-a-go-go related gene (hERG)-type potassium channel blocker, and isoproterenol induced significant changes both in the Ca transient parameters and contractile parameters in a dose-dependent manner. The 3D-hiPSC-CT exhibited doxorubicin-sensitive cytotoxicity and hERG channel blocker/isoproterenol-sensitive electrical activity in vitro, indicating its usefulness for drug-induced cardiotoxicity assays or drug screening systems for drug discovery.


Assuntos
Cardiotoxinas/efeitos adversos , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Contração Muscular/efeitos dos fármacos , Miócitos Cardíacos/patologia , Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxicidade , Sobrevivência Celular , Células Cultivadas , Doxorrubicina/efeitos adversos , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo
14.
Ann Thorac Surg ; 104(5): 1531-1539, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28760462

RESUMO

BACKGROUND: Although implantation of a left ventricular assist device (LVAD) induces reverse remodeling of the left ventricle in end-stage nonischemic dilated cardiomyopathy (DCM), the underlying mechanism is not fully understood. It has been shown that epigenetic modification, such as methylation or acetylation of the histone, is one of the most important upstream signals in cardiac failure. This study hypothesized that histone profiles may be modified by LVAD implantation for end-stage nonischemic DCM, in association with reverse left ventricular remodeling. METHODS: Hemodynamic changes associated with histone modification profiles in the left ventricle were comprehensively assessed in 14 patients with a diagnosis of end-stage nonischemic DCM. These patients underwent LVAD implantation and subsequent cardiac transplantation in our institution (Osaka University Hospital, Osaka, Japan). Samples of normal left ventricle from 3 different people were used as a control. RESULTS: After LVAD support for 2.5 ± 1.2 years, the study cohort showed a significant reverse remodeling of left ventricular function associated with histopathologic changes in the left ventricle, such as reduction of myocyte size. Although the left ventricle of the cohort histologically expressed less 3 histone methylation-related molecules (eg, H3 lysine 4 trimethylation [H3K4me3], H3 lysine 9 dimethylation [H3K9me2], and H3 lysine 9 trimethylation [H3K9me3]) compared with normal left ventricle, LVAD support reversed expression of these molecules, associated with up-regulation of H3 lysine 9 [H3K9] methyltransferase and suppressor of variegation 3-9 homologue 1 [SUV39H1] and with down-regulation of H3K9 demethylase and jumonji domains [JMJDs] in the LVAD-supported left ventricle. Moreover, expression of atrial natriuretic peptide and brain natriuretic peptide (ANP and BNP) was negatively correlated with that of H3K9me2 and H3K9me3. CONCLUSIONS: The epigenetic state of cardiac myocytes (eg, as histone methylation) was substantially modulated in end-stage nonischemic DCM. LVAD support partially reversed the epigenetic state and its upstream signals, in association with pathologic and functional reverse remodeling.


Assuntos
Cardiomiopatia Dilatada/cirurgia , Coração Auxiliar , Código das Histonas/genética , Remodelação Ventricular/genética , Adulto , Idoso , Biópsia por Agulha , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/mortalidade , Cardiomiopatia Dilatada/patologia , Células Cultivadas , Metilação de DNA/genética , Ecocardiografia/métodos , Feminino , Transplante de Coração/métodos , Hemodinâmica/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Peptídeo Natriurético Encefálico/metabolismo , Prognóstico , Medição de Risco , Análise de Sobrevida , Resultado do Tratamento , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/fisiologia
15.
J Radiat Res ; 58(4): 430-438, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28340154

RESUMO

Induced pluripotent stem (iPS) cells have demonstrated they can undergo self-renewal, attain pluripotency, and differentiate into various types of functional cells. In clinical transplantation of iPS cells, however, a major problem is the prevention of tumorigenesis. We speculated that tumor formation could be inhibited by means of irradiation. Since the main purpose of this study was to explore the prevention of tumor formation in human iPS (hiPS) cells, we tested the effects of irradiation on tumor-associated factors such as radiosensitivity, pluripotency and cell death in hiPS cells. The irradiated hiPS cells showed much higher radiosensitivity, because the survival fraction of hiPS cells irradiated with 2 Gy was < 10%, and there was no change of pluripotency. Irradiation with 2 and 4 Gy caused substantial cell death, which was mostly the result of apoptosis. Irradiation with 2 Gy was detrimental enough to cause loss of proliferation capability and trigger substantial cell death in vitro. The hiPS cells irradiated with 2 Gy were injected into NOG mice (NOD/Shi-scid, IL-2 Rγnull) for the analysis of tumor formation. The group of mice into which hiPS cells irradiated with 2 Gy was transplanted showed significant suppression of tumor formation in comparison with that of the group into which non-irradiated hiPS cells were transplanted. It can be presumed that this diminished rate of tumor formation was due to loss of proliferation and cell death caused by irradiation. Our findings suggest that tumor formation following cell therapy or organ transplantation induced by hiPS cells may be prevented by irradiation.


Assuntos
Carcinogênese/patologia , Carcinogênese/efeitos da radiação , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Raios gama , Humanos , Camundongos Endogâmicos NOD , Tolerância a Radiação/efeitos da radiação , Transplante de Células-Tronco
16.
Sci Rep ; 6: 19464, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26763872

RESUMO

Transplantation of induced pluripotent stem cell-derived cardiac tissue constructs is a promising regenerative treatment for cardiac failure: however, its tumourigenic potential is concerning. We hypothesised that the tumourigenic potential may be eliminated by the host immune response after allogeneic cell transplantation. Scaffold-free iPSC-derived cardaic tissue sheets of C57BL/6 mouse origin were transplanted into the cardiac surface of syngeneic C57BL/6 mice and allogeneic BALB/c mice with or without tacrolimus injection. Syngeneic mice and tacrolimus-injected immunosuppressed allogeneic mice formed teratocarcinomas with identical phenotypes, characteristic, and time courses, as assessed by imaging tools including (18)F-fluorodeoxyglucose-positron emission tomography. In contrast, temporarily immunosuppressed allogeneic mice, following cessation of tacrolimus injection displayed diminished progression of the teratocarcinoma, accompanied by an accumulation of CD4/CD8-positive T cells, and finally achieved complete elimination of the teratocarcinoma. Our results indicated that malignant teratocarcinomas arising from induced pluripotent stem cell-derived cardiac tissue constructs provoked T cell-related host immune rejection to arrest tumour growth in murine allogeneic transplantation models.


Assuntos
Transformação Celular Neoplásica , Rejeição de Enxerto/imunologia , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos/patologia , Transplante de Células-Tronco/efeitos adversos , Teratocarcinoma/etiologia , Teratocarcinoma/patologia , Animais , Biópsia , Linhagem Celular , Fluordesoxiglucose F18 , Ordem dos Genes , Vetores Genéticos/genética , Hospedeiro Imunocomprometido , Imunossupressores/administração & dosagem , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/citologia , Tomografia por Emissão de Pósitrons , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tacrolimo/administração & dosagem , Teratocarcinoma/diagnóstico , Transplante Homólogo
17.
Stem Cells Transl Med ; 4(11): 1258-64, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26378261

RESUMO

UNLABELLED: Cell-surface glycans vary widely, depending on cell properties. Previously, we reported that the pattern of N-glycan expression on murine induced pluripotent stem cells (iPSCs) changed toward that of the cardiac tissue during cardiomyogenic differentiation. In this study, N-glycans were isolated from human iPSCs, iPSC-derived cardiomyocytes (iPSC-CMs), and human cardiomyocytes (hCMCs). Their structures were analyzed by a mapping technique based on high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometric data. Of 52 isolated N-glycans, the structures of 38 were clearly identified. In addition, 11 structures were partially identified because the binding style and fucose binding site at the nonreduced terminal could not be identified. Quantitation of each type of N-glycan, based on the terminal glycosylation process, revealed that the exposed N-acetylglucosamine (GlcNAc) and the nonreduced terminal fucose types decreased, whereas the exposed galactose or the α2-3 NeuAc types increased in the iPSCs during cardiomyogenic differentiation. However, the bisecting GlcNAc and the triantennary structures were found in relative abundance in the iPSC-CMs in comparison with hCMCs or iPSCs. Expression of MGAT3, a glycosyltransferase-encoding gene that produces the bisecting GlcNAc structures, was higher in iPSCs and iPSC-CMs than in hCMCs. These findings will prove useful in understanding the directional precision of cardiomyogenic differentiation in vitro. SIGNIFICANCE: This study focused on N-glycans produced in human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes to investigate their change on cardiomyogenic differentiation in vitro. This shows that the expression pattern of N-glycans in human iPSCs changed toward the pattern observed in human cardiomyocytes upon cardiomyogenic differentiation. Structural differences were also observed in the bisecting N-acetylglucosamine and the triantennary structures upon cardiomyogenic differentiation. The findings of this study will help in understanding the directional precision of cardiomyogenic differentiation in vitro.


Assuntos
Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , N-Acetilglucosaminiltransferases/biossíntese , Polissacarídeos/biossíntese , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , N-Acetilglucosaminiltransferases/genética , Polissacarídeos/genética
18.
PLoS One ; 9(10): e111064, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25357199

RESUMO

Cell surface glycans vary widely, depending on cell properties. We hypothesized that glycan expression on induced pluripotent stem cells (iPSCs) might change during cardiomyogenic differentiation toward the myocardial phenotype. N-glycans were isolated from iPSCs, iPSC-derived cardiomyocytes (iPSC-CM), and original C57BL/6 mouse myocardium (Heart). Their structures were analyzed by a mapping technique based on HPLC elution times and MALDI-TOF/MS spectra. Sixty-eight different N-glycans were isolated; the structures of 60 of these N-glycans were identified. The quantity of high-mannose type (immature) N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart. We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans. Some structural differences were detected between iPSC-CM and Heart. No N-glycolyl neuraminic acid (NeuGc) structures were detected in iPSC-CM, whereas the heart contained numerous NeuGc structures, corresponding to the expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase. Furthermore, several glycans containing Galα1-6 Gal, rarely identified in the other cells, were detected in the iPSC-CM. The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures. Further studies will be warranted to reveal the meaning of the difference of N-glycans between the iPSC-CM and the myocardium.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Polissacarídeos/metabolismo , Animais , Configuração de Carboidratos , Linhagem Celular , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Miócitos Cardíacos/citologia
20.
Circulation ; 128(11 Suppl 1): S87-94, 2013 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-24030425

RESUMO

BACKGROUND: Transplantation of cardiomyocytes that are derived from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) shows promise in generating new functional myocardium in situ, whereas the survival and functionality of the transplanted cells are critical in considering this therapeutic impact. Cell-sheet method has been used to transplant many functional cells; however, potential ischemia might limit cell survival. The omentum, which is known to have rich vasculature, is expected to be a source of blood supply. We hypothesized that transplantation of hiPS-CM cell sheets combined with an omentum flap may deliver a large number of functional hiPS-CMs with enhanced blood supply. METHODS AND RESULTS: Retrovirally established human iPS cells were treated with Wnt signaling molecules to induce cardiomyogenic differentiation, followed by superparamagnetic iron oxide labeling. Cell sheets were created from the magnetically labeled hiPS-CMs using temperature-responsive dishes and transplanted to porcine hearts with or without the omentum flap (n=8 each). Two months after transplantation, the survival of superparamagnetic iron oxide-labeled hiPS-CMs, assessed by MRI, was significantly greater in mini-pigs with the omentum than in those without it; histologically, vascular density in the transplanted area was significantly greater in mini-pigs with the omentum than in those without it. The transplanted tissues contained abundant cardiac troponin T-positive cells surrounded by vascular-rich structures. CONCLUSIONS: The omentum flap enhanced the survival of hiPS-CMs after transplantation via increased angiogenesis, suggesting that this strategy is useful in clinical settings. The combination of hiPS-CMs and the omentum flap may be a promising technique for the development of tissue-engineered vascular-rich new myocardium in vivo.


Assuntos
Sobrevivência Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Transplante de Células-Tronco Mesenquimais/métodos , Miócitos Cardíacos/transplante , Retalhos Cirúrgicos/fisiologia , Regulação para Cima/fisiologia , Animais , Feminino , Coração/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Distribuição Aleatória , Suínos , Porco Miniatura
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