Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method / La eficiencia de la expresión transg é nica en células bovinas varía según el tipo de célula y el método de la transferencia de genes / A eficiência da expressão do transgene em células bovinas varia de acordo com o tipo celular e o método de transferência gênica

Abstract Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.

Resumen Antecedentes: La producción de animales transgénicos sigue siendo una biotecnología de baja eficiencia, y se deberían utilizar alternativas sencillas para mejorar la tasa de producción de bovinos transgénicos mediante transferencia nuclear. Una de estas alternativas es la selección del tipo mas apropiado de célula donante y método de transferencia génica. Objetivo: Investigar el efecto del tipo celular (fibroblastos fetales o adultos, y celulas del cumulus), y el método de transferencia génica (lipofección y transducción lentiviral) en la incorporación, expresión génica, y la intensidad de fluorescencia del transgén en células bovinas analizadas por citometría de flujo. Métodos: Fibroblastos fetales (FF), fibroblastos adultos (AF), y células del cúmulo (CC) fueron transfectados a través de lipofección o transducidos utilizando partículas lentivirales producidas con plásmidos que expresan la proteína verde fluorescente (GFP). Resultados: La transducción lentiviral dio lugar a mayores tasas de expresión del transgen en todos los tipos de células (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96, CC: 60,7% ± 14,7) en comparación con la lipofección (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). Las células del cúmulus mostraron menores tasas de expresión del transgen que los otros tipos celulares. En cuanto a la intensidad de fluorescencia, no hubo diferencias significativas entre lipofección y transducción lentiviral; en ambos tratamientos, se obtuvo una mayor intensidad de fluorescencia cuando se usaron células adultas en lugar de células fetales. Conclusión: La eficiencia de la transferencia de genes varía según el tipo de célula y el método de transferencia génica, con la transducción lentiviral se logra una mayor tasa de transfección, y los fibroblastos adultos muestran una mejor expresión transgénica.

Resumo Antecedentes: A produção de animais transgênicos é uma biotecnologia que ainda apresenta baixa eficiência e alternativas simples devem ser usadas para o aumento da produção de bovinos transgênicos por transferência nuclear. Uma destas alternativas compreende a seleção do tipo apropriado de célula doadora de núcleo e do método de transferência gênica. Objetivo: Investigar a influência do tipo celular (fibroblastos fetais ou adultos, e células do cumulus), e do método de transferência gênica (transfecção por lipofecção ou transdução lentiviral) na incorporação, expressão, e na intensidade de fluorescência do transgene em células bovinas analisadas por citometria de fluxo. Métodos: Fibroblastos fetais (FF), fibroblastos adultos (AF), e células do cumulus (CC) foram submetidas à lipofecção ou à transfecção lentiviral utilizando plasmídeos expressando a Proteína Fluorescente Verde - GFP). Resultados: A transdução lentiviral resultou em maiores taxas de expressão do transgene em todos os tipos celulares (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96; CC: 60,7% ± 14.7) quando comparada com a lipofeccção (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). As células do cumulus apresentaram menores taxas de expressão quando comparadas aos outros tipos celulares. Com relação à intensidade de fluorescência, não houve diferença significativa entre a lipofecção e a transdução lentiviral e em ambos os tratamentos as células adultas apresentaram maior intensidade de fluorescência do que as células fetais. Conclusão: A eficiência de transferência gênica varia de acordo com o tipo celular, e com o método de transferência gênica, sendo que a transdução lentiviral resultou em maiores taxas, e que os fibroblastos adultos mostraram melhor expressão do transgene.

Studies on the long non-coding RNA during the reprogramming of human pluripotent stem cells / 天津医药

Objective To investigate the changes and roles of the long non-coding RNA (IncRNA)during the reprogramming of human induced pluripotent stem cells. Methods Agilent Human lncRNA (4 × 180K) chip was used to check the expression of lncRNA in somatic cells, induced pluripotent stem cells and embryonic stem cells. Compared with differentially expressed lncRNA in somatic cells and induced pluripotent stem cells, lncRNA was selected that may play an important role during the reprogramming of human pluripotent stem cells. Results The lncRNA expression profiles in induced pluripotent stem cells were similar to embryonic stem cells, but were different from the somatic cells. A total of 3 156 differentially expressed lncRNAs were found between stem cells and somatic cells by cluster analysis, and 222 differentially expressed lncRNAs were found during the reprogramming process of human pluripotent stem cells by biological analysis. Conclusion lncRNA may play an important role in reprogramming process of human pluripotent stem.

Uso de células-tronco pluripotentes induzidas para compreensão de alterações em cardiomiócitos de pacientes com cardiomiopatias de base-genética / Induced pluripotent stem cells to study cardiomyocytes derived from patients with genetic cardiomyopathies

O estudo de mutações genéticas como causa das cardiomiopatias teve início com a descoberta de mutações em proteínas sarcoméricas que levavam à Cardiomiopatia Hipertrófica, desde então, alterações em diversos genes, de proteínas contráteis ou não, foram descobertas e listadas como a responsável pelo desenvolvimento de diferentes cardiomiopatias. Estudar o efeito destas mutações nos cardiomiócitos destes pacientes permanecia um desafio devido ao difícil acesso às células cardíacas. Em 2007, a técnica de reprogramação de células somáticas em células-tronco pluripotentes foi descoberta. Pelo fato das células-tronco pluripotentes serem capazes de ser diferenciadas em cardiomiócitos, surgiu-se a possibilidade de se estudar essas células de indivíduos portadores das mutações genéticas. Esta tese teve como objetivo a criação de um modelo celular para estudar a Cardiomiopatia Hipertrófica causada por mutações genéticas. Inicialmente foi estabelecido um protocolo de reprogramação celular para se estabelecer linhagens celulares das células-tronco induzidas de um paciente com mutação no gene MYH7. Tendo as células caracterizadas, elas foram diferenciadas em cardiomiócitos através de um protocolo adaptado de protocolos de diferenciação direta em cardiomiócitos. Os cardiomiócitos gerados apresentaram características moleculares e funcionais semelhantes à cardiomiócitos primários humanos e foi visualizado, através de microscopia eletrônica de transmissão, que os cardiomiócitos do paciente com alteração genética possuíam grande proporção de sarcômeros desorganizados em comparação a cardiomiócitos de indivíduos saudáveis. Em conclusão, o modelo celular desenvolvido sugere ser possível o estudo do efeito de mutações genéticas em Cardiomiopatia Hipertrófica.

The study of genetic mutations as the cause of cardiomyopathies initiates with the discovery of mutations in sarcomeric proteins genes that lead to Hypertrophic Cardiomyopathy. Since then, mutations in several genes, coding to sarcomeric proteins or not, were discovered and listed as the reason to the cardiomyopathies. To study the effect of these mutations was a challenge due the difficulty to accesses cardiac cells. In 2007, the technique of reprogramming somatic cells into pluripotent stem cells was discovered. The fact that the pluripotent stem cells are capable of differentiating into cardiomyocytes opened the opportunity to study these cells from individuals with genetic mutations. This thesis aimed to create a cellular model to study Hypertrophic Cardiomyopathy caused by genetic mutations. Initially we established a cell reprogramming protocol to establish induced stem cells lines from a patient with mutation in MYH7 gene. Having characterized the cells, they were differentiated into cardiomyocytes using an adapted protocol from direct differentiation protocols. Cardiomyocytes generated showed molecular and functional characteristics similar to human primary cardiomyocytes and were visualized by means of transmission electron microscopy. The patient's cardiomyocytes had a large proportion of disorganized sarcomeres compared to cardiomyocytes from healthy individuals. In conclusion, the cell model developed suggests that it is possible to study the effect of genetic mutation in Hypertrophic Cardiomyopathy using induced pluripotent stem cells derived cardiomyocytes.

Descrubrimientos sobre reprogramación nuclear merecen el premio Nobel en 2012 / Discoveries on nuclear reprogramming deserves the Nobel prize in 2012

Este artículo fue escrito para honrar a J.B Gurdon y S. Yamanaka, laureados con el Premio Nobel en Fisiología p Medicina 2012 "por el descubrimiento de que las células maduras pueden ser reprogramadas para volverse pluripotentes". Se presentan en forma concisa sus aportes científicos y reseñas biográficas. J.B. Gardon, en Inglaterra, demostró hace 50 años en anfibios que al trasplantar el núcleo de una célula intestinal a un huevo u ovocito enuncleado se obtiene una célula totipotente que se convierte en un embrión y se desarrolla hasta convertirse en una rana adulta, lo cual implica la conservación de genoma en el proceso de diferenciación y la resersibilidad de dicho proceso. Estos descubrimientos llevaron a que otros autores realizaran la clonación de mamiferos utilizando el núcleo de células somáticas y la obtención de células madre pluripotentes a partir de los embrines que se producen in vitro por el desarrollo de las células totipotentes. Se mencionan varias aplicaciones y las contribuciones de Gurdon para comprender el proceso de reprogramación. S. Yamanaka, en Japón, hace seis años, reprogramó al estado embrionario fibroblastos cutáneos de ratones y humanos adultos insertando mediante vectores retrovirales una combinación de los genes de cuatro factores de transcripción: Oct3/4, Sox2, Klf4 y c-Myc. Las células reprogramadas fueron denominadas células madre pluripotentes inducidas. Utilizando la técnica desarrollada por Yamanaka y otras surgidas a raiz de sus descrubrimientos, miles de personas obtienen ahora células madre pluripotentes inducidas a partir de muchas especies y tejidos, incluyendo seres humanos sanos y enfermos. Las células madre pluripotentes o sus derivadas tienen un amplio potencial de aplicación, entre ellas, estudios de embriología y fisiopatología, modelos de enfermedades, descubrimiento de drogas y terapias celulares

This paper was written to honor J.B Gurdon y S. Yamanaka, 2012 Nobel Prize laureates in Physiology or Medicine for "the discovery that mature cells can be reprogrammed to become pluripotent". Their main scientific contributions and biography are presented in a concise manner. JB Gurdon, in England, showed fifty years ago in amphibians that the transplantation of the nucleus of an intestinal cell to an enucleated egg or oocyte produces a totipotent cell that develops into an embryo and adult frog. This implies that cellular differentiation is reversible and the genome is conserved in that process. The discoveries led to the cloning of mammals by other authors using the nucleus of somatic cells and to obtain pluripotent stem cells in vitro from the embryos produced by development of the totipotent cells. Some applications are considered. Gurdon's contribution to the understanding of the reprogramming process is mentioned. S. Yamanaka six years ago in Japan reprogrammed skin fibroblastis from adult mice and humans to the embryonic state by introducing via retroviral vectors a combination of the genes of 4 transcription factors, Oct3/4. Sox2, Klf4 and c-Myc. The reprogammed cells were named induced pluripontent stem cells. Throusands of people are now producing induced pluripotent stem cells from many tissues and species, including healthy and ill humans, using Yamanaka's methods and other techniques stimulated by his work. Pluripotent stem cells or their derivatives have great potential for a wide range of applications including research in embryology and pathophysiology, disease modeling, drug discovery and cell transplantation therapies

Modeling of Human Genetic Diseases Via Cellular Reprogramming

The generation of induced pluripotent stem cells (iPSCs) derived from patients' somatic cells provides a new paradigm for studying human genetic diseases. Human iPSCs which have similar properties of human embryonic stem cells (hESCs) provide a powerful platform to recapitulate the disease-specific cell types by using various differentiation techniques. This promising technology has being realized the possibility to explore pathophysiology of many human genetic diseases at the molecular and cellular levels. Furthermore, disease-specific human iPSCs can also be used for patient-based drug screening and new drug discovery at the stage of the pre-clinical test in vitro. In this review, we summarized the concept and history of cellular reprogramming or iPSC generation and highlight recent progresses for disease modeling using patient-specific iPSCs.

Modeling of Human Genetic Diseases Via Cellular Reprogramming

The generation of induced pluripotent stem cells (iPSCs) derived from patients' somatic cells provides a new paradigm for studying human genetic diseases. Human iPSCs which have similar properties of human embryonic stem cells (hESCs) provide a powerful platform to recapitulate the disease-specific cell types by using various differentiation techniques. This promising technology has being realized the possibility to explore pathophysiology of many human genetic diseases at the molecular and cellular levels. Furthermore, disease-specific human iPSCs can also be used for patient-based drug screening and new drug discovery at the stage of the pre-clinical test in vitro. In this review, we summarized the concept and history of cellular reprogramming or iPSC generation and highlight recent progresses for disease modeling using patient-specific iPSCs.

Cellular reprogramming of somatic cells.

The process of ‘cell reprogramming’ can be achieved by somatic cell nuclear transfer, cell fusion with embryonic stem cells, exposure to stem cell extracts, or by inducing pluripotentcy mediated by defined factors giving rise to what are termed induced pluripotent stem cells. More recently, the fate of a somatic cell can be directly induced to uptake other cell fates, termed lineage-specific reprogramming, without the need to de-differentiate the cells to a pluripotent state. In this review we will describe the different methods of reprogramming somatic cells.

Induced pluripotent stem cells and their application in research and treatment of cerebrovascular disease / 国际脑血管病杂志

Induced pluripotent stem (iPS) cells are reprogrammed by the differentiated adult cells, ard in terms of biological characteristics they are remarkably similar to embryonic stem cells. Compared to the embryonic stem cells, the iPS cells are unrestricted by cell resources, immune rejection and ethics, They maintain the advantages such as specific individual genes, and provide potential cell resources for the fields of regenerative medicine and tissue engineering, This article mainly introduces the iPS cells and their application in the research and treatment of cerebrovascular diseases.

Reprogramación nuclear y células pluripotentes inducidas / Nuclear reprogramming and induced pluripotent cells / Reprogramação nuclear e células pluripotentes induzidas

La reprogramación de células somáticas para generar células madre pluripotentes inducidas (iPS), ha sido uno de los avances más importantes de la biología en los últimos años. La identificación de un grupo de factores de transcripción y más recientemente de algunos compuestos químicos que pueden inducir la pluripotencia en células somáticas, brinda una oportunidad única para el estudio de los mecanismos celulares y moleculares de la diferenciación celular y promete la posibilidad de generar células pluripotentes paciente-específicas para el tratamiento de múltiples enfermedades en protocolos terapia celular y medicina regenerativa...

Reprogramming of somatic cells to generate induced pluripotent stem cells (iPS), has been one of the most important advances in biology in recent years. The identification of a group of transcription factors and more recently of some chemical compounds that can induce pluripotency in somatic cells provides a unique opportunity to study cellular and molecular mechanisms of cell differentiation and promises the possibility of generating patient-specific pluripotent stem cells for the treatment of multiple diseases in protocols of cell therapy and regenerative medicine...

A reprogramação de células somáticas para gerar células-tronco pluripotentes induzidas (iPS), tem sido um dos mais importantes avanços na biologia nos últimos anos. A identificação de um conjunto de fatores de transcrição e, mais recentemente, de alguns compostos químicos que podem induzir pluripotência em células somáticas produzem uma oportunidade única para estudar os mecanismos celulares e moleculares da diferenciação celular e promete a capacidade de gerar células pluripotentes doente-específicas para o tratamento de doenças múltiplas nos protocolos de terapía celular e medicina regenerativa...