RESUMO
BACKGROUND: Lack of evidence in literature to show low vision care enhances the reading performance in children with Multiple Disabilities and Visual Impairment (MDVI). AIM: To evaluate the effectiveness of Low Vision Care intervention on the reading performance of children with MDVI. MATERIALS AND METHODS: Three subjects who were diagnosed to have cerebral palsy and visual impairment, studying in a special school were recruited for the study. All of them underwent detailed eye examination and low vision care evaluation at a tertiary eye care hospital. A single subject multiple baseline (study) design was adopted and the study period was 16 weeks. The reading performance (reading speed, reading accuracy, reading fluency) was evaluated during the baseline phase and the intervention phase. The median of all the reading parameters for each week was noted. The trend of the reading performance was graphically represented in both the phases. RESULTS: Reading speed increased by 37 Word per minute, 37 Letters per minute and 5 letters per minute for the subject 1, 2 and 3 respectively after the intervention. Reading accuracy was 84%, 91% and 86.4% at the end of the baseline period and 98.7%, 98.4% and 99% at the end of 16 weeks for subject 1, 2 and 3 respectively. Average reading fluency score was 8.3, 7.1 and 5.5 in the baseline period and 10.2, 10.2 and 8.7 in the intervention period. CONCLUSION: This study shows evidence of noticeable improvement in reading performance of children with MDVI using a novel study design.
Assuntos
Cegueira/reabilitação , Atenção à Saúde/métodos , Pessoas com Deficiência/reabilitação , Movimentos Oculares/fisiologia , Leitura , Baixa Visão/reabilitação , Acuidade Visual/fisiologia , Adolescente , Criança , Pré-Escolar , Seguimentos , Humanos , Masculino , Inquéritos e Questionários , Baixa Visão/fisiopatologiaRESUMO
AIM: Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study, we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS: hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture, and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition, the cell growth and differentiation process was adaptable to large culture formats. Moreover, the cardiomyocytes survived following cryopreservation, and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION: These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium, a step forward towards the application of these cells to human clinical use or drug discovery.
Assuntos
Criopreservação/métodos , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Meios de Cultura Livres de Soro , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RatosRESUMO
Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.
Assuntos
Células-Tronco Embrionárias/citologia , Sobrevivência de Enxerto , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Animais , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Ecocardiografia , Ventrículos do Coração/metabolismo , Humanos , Imageamento por Ressonância Magnética , Masculino , Miócitos Cardíacos/transplante , Ratos , Ratos Sprague-DawleyRESUMO
Human embryonic stem (hES) cell-derived cardiomyocytes hold great promise for cardiovascular regenerative medicine. However, this application faces a number of challenges, including generating cardiomyocytes of adequate purity. With current protocols being used by several laboratories, cardiomyocyte differentiation from hES cells occurs at low frequency and results in a mixture of differentiated cells. Here we describe a novel method for enrichment of cardiomyocytes. Cardiomyocytes were isolated from embryoid body (EB) outgrowths by Percoll separation and then enriched by culturing the aggregates of cells (termed cardiac bodies, CBs) in suspension. The majority of CBs showed contractility after 1 week in culture and were positive for multiple cardiomyocyte- associated proteins. Enrichment of cardiomyocytes was evident by the increase in the expression of cardiac alpha and beta myosin heavy chains (alpha and betaMHC) in CBs in suspension culture compared to unpurified EB outgrowths. Flow cytometry analysis showed that 35-66% of the cells in CBs were positive for sarcomeric myosin heavy chain (sMHC) or cardiac troponin T (cTnT) expression. In addition, dissociated CBs were capable of reassociating into contracting aggregates in suspension and recovering contractility after the individual cells were replated onto matrix-coated surfaces. These data suggest that the CB method is a useful approach for the generation of cardiomyocytes at an adequate purity for cardiovascular therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Linhagem da Célula , Separação Celular , Citometria de Fluxo , Expressão Gênica , HumanosRESUMO
Current procedures for the maintenance of cardiomyocytes from human embryonic stem (hES) cells rely on either co-culture with mouse cells or medium containing fetal bovine serum (FBS). Due to exposure to animal products, these methods carry the risk of potential pathogen contamination and increased immunogenicity. Additionally, FBS introduces inherent variability in the cultures due to the inevitable differences in serum lots. Here we investigated whether a defined serum-free medium containing creatine, carnitine, taurine, and insulin (CCTI) could maintain hES cell-derived cardiomyocytes. We show that hES cell-derived cardiomyocytes maintained in the CCTI medium in the absence of any feeders exhibit similar phenotypes to those maintained in serum, as indicated by the following observations: (1) comparable levels of cardiac gene transcription were found in cells grown in serum-containing medium versus those in the CCTI medium; (2) cardiomyocyte-associated proteins were expressed in cells cultured in the CCTI medium; (3) beating cells in the CCTI medium responded to pharmacological agents in a dose-dependent manner; and (4) the vast majority of the beating embryoid bodies displayed ventricular-like action potentials (APs), and the ventricular cells in serum-containing medium and the CCTI medium had indistinguishable AP properties. Therefore, culturing hES cell-derived cardiomyocytes in serum-free medium as described here should facilitate the use of the cells for in vitro and in vivo applications.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Coração/fisiologia , Células Musculares/fisiologia , Miocárdio/citologia , Carnitina , Técnicas de Cultura de Células/métodos , Creatina , Meios de Cultura Livres de Soro , Células-Tronco Embrionárias/fisiologia , Humanos , Insulina , Células Musculares/citologia , TaurinaRESUMO
Human embryonic stem cells (hESCs) offer the opportunity to replenish cells lost in the postinfarct heart. We explored whether human myocardium could be generated in rat hearts by injecting differentiated cardiac-enriched hESC progeny into the left ventricular wall of athymic rats. Although initial grafts were predominantly epithelial, noncardiac elements were lost over time, and grafts consisted predominantly of cardiomyocytes by 4 weeks. No teratomatous elements were observed. Engrafted cardiomyocytes were glycogen-rich and expressed expected cardiac markers including beta-myosin heavy chain, myosin light chain 2v, and atrial natriuretic factor. Heat-shock treatment improved graft size approximately threefold. The cardiac implants exhibited substantial angiogenesis, both recipient and graft derived. Importantly, there was greater proliferation in human cardiomyocytes than previously seen in rodent-derived cardiomyocytes: 14.4% of graft cardiomyocytes expressed the proliferation marker Ki-67, and 2.7% incorporated the thymidine analog BrdU 4 weeks after transplantation. This proliferation was associated with a sevenfold increase in graft size over the 4-week interval. Thus, hESCs can form human myocardium in the rat heart, permitting studies of human myocardial development and physiology and supporting the feasibility of their use in myocardial repair.
Assuntos
Procedimentos Cirúrgicos Cardíacos , Diferenciação Celular , Miócitos Cardíacos/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Transplante Heterólogo , Animais , Divisão Celular , Temperatura Alta , Humanos , Masculino , Miócitos Cardíacos/fisiologia , Neovascularização Fisiológica , Ratos , Ratos Nus , Células-Tronco/fisiologia , Fatores de TempoRESUMO
Cell replacement therapy is a promising approach for the treatment of cardiac diseases, but is challenged by a limited supply of appropriate cells. We have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem (hES) cells. Cardiomyocyte differentiation was evaluated using 3 parent (H1, H7, and H9) hES cell lines and 2 clonal (H9.1 and H9.2) hES cell lines. All cell lines examined differentiated into cardiomyocytes, even after long-term culture (50 passages or approximately 260 population doublings). Upon differentiation, beating cells were observed after one week in differentiation conditions, increased in numbers with time, and could retain contractility for over 70 days. The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac alpha-myosin heavy chain, cardiac troponin I and T, atrial natriuretic factor, and cardiac transcription factors GATA-4, Nkx2.5, and MEF-2. In addition, cardiomyocyte differentiation could be enhanced by treatment of cells with 5-aza-2'-deoxycytidine but not DMSO or retinoic acid. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll density centrifugation to give a population containing 70% cardiomyocytes. The enriched population was proliferative and showed appropriate expression of cardiomyocyte markers. The extended replicative capacity of hES cells and the ability to differentiate and enrich for functional human cardiomyocytes warrant further development of these cells for clinical application in heart diseases.