A tecnologia time-lapse pode predizer a qualidade embrionária antes dos resultados do PGD (diagnósticogenético pré-implantacional)? / Technology time-lapse can predict embryo quality of results before PGD(pre-implantation genetic diagnosis)?

Objetivo: Verificar se há correlação entre os resultados obtidos via diagnóstico genético préimplantacional (PGD) e os dados obtidos via monitoramento em tempo real (time-lapse) durante os três dias de desenvolvimento embrionário. Método: Estudo retrospectivo no qual foram avaliados embriões de ciclos de injeção intracitoplasmática de espermatozóides (ICSI) de março a junho de 2012. Após a ICSI, os embriões foram colocados em incubadora vertical com monitoramento time-lapse durante três dias. A seguir os embriões foram submetidos ao PGD. Os resultados obtidos foram baseados na análise do tempo de aparecimento e desaparecimento dos dois pró-núcleos, de início da primeira e da segunda clivagem e de intervalo entre esses eventos, além da observação dos corpúsculos polares. Resultados: Dados mostraram que 84,6% dos embriões apresentaram um padrão de ciclo celular assincrônico e a inviabilidade comprovada com os resultados do PGD, 7,7% dos embriões considerados normais nos resultados do PGD mostraram ter ciclos celulares fora dos padrões e 7,7% dos embriões com ciclo celular normal apresentaram ao PGD anomalias múltiplas. As diferenças foram estatisticamente significantes para p < 0,05. Embriões com PGD normal podem apresentar um ciclo celular assincrônico, o que afeta sua implantação. Conclusões: O estudo preliminar mostra que os dados obtidos com a metodologia time-lapse, em primeiro momento, podem ser usados para avaliar a qualidade embrionária em conjunto com a avaliação morfológica deles, independentemente dos resultados do PGD, pois alterações no padrão de desenvolvimento celular embrionário parecem afetar a implantação, salvo algumas exceções. Porém, como o N amostral ainda é pequeno, necessita-se de um período maior para a certificação dos eventos observados.

Objective: Investigate whether there is correlation between results obtained with PGD and data obtained monitoring “time lapse” during 3 days of embryonic development. Method: This is a retrospective study in which we assessed embryos ICSI cycles from March to December 2012. Upon completion ICSI, embryos were placed in an incubator with vertical monitoring “time lapse” for 3 days, then embryos were subjected to PGD. The results were based on analysis of the time of appearance of two pronuclei, disappearance, the start time of first and second cleavage and intervals between these events, and the observation of polar bodies. Results: Data show that 84.6% of the embryos exhibited a pattern of cell cycle asynchronous and viability confirmed by the results of PGD, and 7.7% of embryos considered normal results of PGD, proved to have cell cycle asynchronous, 7.7% of embryos with normal cell cycle PGD showed multiple anomalies, differences were statistically significant at p < 0.05. PGD embryos with normal can present asynchronous cell cycle, affecting its implantation. Conclusion: The preliminary study shows that data obtained with method “time-lapse”, can be used to evaluate the embryo quality together with morphological evaluation same regardless outcome of PGD, because changes in pattern development of embryonic cell seem to affect development, with some exceptions. However, such as sample size remains small needs to be a longer period for certification of observed events.

Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP

OBJECTIVE: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed andconfirmed by PCR and digesti on analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR.CONCLUSION: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.

La bioetica y la dignidad humana Que hay de nuevo sobre la Ética y el Pre-embrión Humano? / Bioethics and human dignity

El reconocimiento universal a la dignidad humana no ha sido una empresa fácil de conseguir ni tampoco es comprensible para muchos contemporáneos. Las razones son muchas, pero sobresalen estas:Necesidad de un desarrollo del juicio moral que haga comprensible los valores morales universales.Desarrollo incipiente de sociedades bien organizadas.Climas políticos propios.También se necesita dilucidar “por qué las personas son dignas”, entendiendo por dignidad una cualidad transitiva que implica que alguien es merecedor de recibir un trato especial a cambio o en proporción a un mérito o condición también especial. Lo anterior nos pone a pensar sobre la siguiente afirmación: “El pre-embrión humano no es persona, pero sí el embrión. El pre-embrión humano es persona humana potencial”.Entonces...¿será el pre-embrión un ser carente de dignidad humana?.

effect of voltage and duration of electrical stimulus on fusion and cleavage of electrofused two-cell bovine embryos

The value of embryonic stem-like cells in cloning is obvious. Production of cloned animals can be achieved by introduction of these cells into a tetraploid embryo. Tetraploid embryo is used as a feeder for development of embryonic stem-like cells and can be produced in vitro by electro-fusion of 2-cell embryos. The aim of this study was to assess the effect of voltage alteration and duration on fusion and cleavage rates of bovine tetraploid embryo produced by electrofusion. After in vitro maturation and fertilization of cumulus oocyte complexes, two-cell embryos were categorized into three groups: 1- Fused group [FG]: include two-cell embryos fused by exposure to different voltages [0.5, 0.75, 1, 1.25 and 1.5 kV/cm] and durations [20, 40, 60, 80 and 100 micro s]. 2- Exposed control group [ECG]: two-cell embryos that remained unfused after electrofusion. 3- Unexposed control group [UCG]: two-cell embryos cultured without exposure to any voltage. The embryos from each group were cultured in SOF1. The fusion and cleavage rates were compared in each group. Increase in voltage resulted in significantly higher fusion and lower cleavage rate. The increased duration had no significant effect on fusion rate. The increased duration of high voltages caused decreased cleavage rate significantly, and in low voltage resulted in increased cleavage rate. The cleavage rate in ECG group followed the same as FG group, and they were lower when compared to UCG. Best fusion and cleavage rate was obtained at 0.75-1 kV/cm for 60 micro s duration

Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.

Human embryonic stem cells and therapeutic cloning

The remarkable potential of embryonic stem (ES) cells is their ability to develop into many different cell types. ES cells make it possible to treat patients by transplanting specialized healthy cells derived from them to repair damaged and diseased cells or tissues, known as "stem cell therapy". However, the issue of immunocompatibility is one of considerable significance in ES cell transplantation. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from nuclear transfer of the patient's own cells. This concept is known as "therapeutic cloning". In this review, we describe the derivations of ES cells and cloned ES cells by somatic cell nuclear transfer, and their potential applications in transplantation medicine.

Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells

Human embryonic stem cells (hESCs) need feeder cells for their maintenance in an undifferentiated state. In conventional culture systems, mouse embryonic fibroblasts (MEFs) serve as feeder cells to maintain hESCs. However, the use of MEFs elevates the risk of transmitting mouse pathogens and thus limits the potential of hESCs in cell replacement therapy. Consequently, the use of human feeder cells would be an important step forward in this in vitro technology. To address this issue, we used fibroblast-like cells differentiated from the Miz-hES6 hESC line (Diff (Miz-hES6)) as feeder cells to support the in vitro growth of three hESC lines. Immunofluorescence microscopy and reverse transcription-PCR assessing the expression of undifferentiated hESC markers revealed all three hESC lines were maintained in an undifferentiated state. In vitro proliferation proceeded as efficiently as when the hESCs were cultured on MEFS. Moreover, karyotype analysis revealed the chromosomal normality of the hESC lines and the Diff (Miz-hES6) feeders themselves after even 50 passages. Furthermore, the hESC lines maintained their pluripotency since they remained capable of forming embryoid bodies (EBs) in vitro. Thus, hESC-derived fibroblast-like cells successfully support in vitro hESC propagation.

Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.

Hematopoietic Differentiation of Embryoid Bodies Derived from the Human Embryonic Stem Cell Line SNUhES3 in Co-culture with Human Bone Marrow Stromal Cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.

Tissue expression of platelet endothelial cell adhesion molecule-1 at pre and postnatal murine development

Endothelial cells, at the cell-cell borders, express PECAM-1, and have been implicated in vascular functions. The monoclonal antibody MEC 13.3 recognizers PECAM-1 molecule from mouse vessels and allows to analyse the ontogeny of mouse endothelium. At the present, little is known about the molecular basis of differentiation pathways of endothelial cells, that enables its morphological heterogeneity. The purpose of this study was to analyze the pattern of PECAM-1 expression, employing monoclonal antibody MEC 13.3, in cellular suspensions obtained from different mouse organs at pre and postnatal stages. Fluorescence activated cell sorter analysis showed a different profile of the glycoprotein expression in a cell population with size and granularity selected by 1G11 endothelial cell line. The expression differs from prenatal to postnatal developmental stages in a given organ, and among the organs studied. Another cell population, with a size and granularity higher than 1G11 endothelial cell line, coexists in cellular suspensions obtained from liver, gut and brain. These cells could be related to those detected by means of immunoenzyme methods which showed a non-differentiated morphology. The different PECAM-1 pattern expression could reflect potential organ-specific differentiation pathways during development and according to organs environment. The existence of another cell population with a size and granularity higher than 1G11 endothelial cell line required a phenotypic characterization.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.

5-azacytidine induces cardiac differentiation of P19 embryonic stem cells

The P19 embryonal carcinoma cell line is a useful model cells for studies on cardiac differentiation. However, its low efficacy of differentiation hampers its usefulness. We investigated the effect of 5-azacytidine (5-aza) on P19 cells to differentiate into a high-efficacy cardiomyocytes. Embryoid-body-like structures were formed after 6 days with 1 micrometer of 5-aza in a P19 cell monolayer culture, beating cell clusters first observed on day 12, and, the production of beating cell clusters increased by 80.1% (29 of 36-wells) after 18 days. In comparison, the spontaneous beating cells was 33.3% (12 of 36-wells) for the untreated control cells. In response to 1 micrometer of 5-aza, P19 cells expressed bone morphogenetic protein-2 (BMP-2), BMP-4, Bmpr1a and Smad1 at day 6 or 9, and also cardiac markers such as GATA-4, Nkx2.5, cardiac troponin I, and desmin were up-regulated in a time-dependent manner after induction of BMP signaling molecules. Immunocytochemistry revealed the expression of smooth muscle a-actin, sarcomeric a-actinin, cardiac myosin heavy chain, cardiac troponin T and desmin, respectively. The proportion of sarcomeric a-actinin positive cells accounted for 6.48% on day 15 after 5-aza exposure as measured by flow cytometry. This study has demonstrated that 5-aza induces differentiation of P19 cells into cardiomyocytes in a confluent monolayer culture in the absence of prior embryoid formation and dimethyl sulfoxide exposure, depending in part on alteration of BMP signaling molecules. These results suggest that 5-aza treatment could be used as a new method for cardiac differentiation in P19 cells.

Comparaçäo entre três escores embrionários como fator prognóstico do sucesso em fertilizaçäo in vitro / Comparison between three embryo scoring systems as predictive of the outcome of in vitro fertilization

OBJETIVO: avaliar três sistemas de escore embrionário para embriöes de 3º dia e correlacioná-los com resultados positivos da técnica de fertilizaçäo in vitro. MÉTODO: estudo retrospectivo desenvolvido pelo programa de fertilizaçäo in vitro do Hospital das Clínicas da Faculdade de Medicina de Ribeiräo Preto - USP. Foram incluídas 137 pacientes submetidas a fertilizaçäo in vitro e transferência de 439 embriöes. Os principais resultados avaliados foram taxa de gravidez e taxa de implantaçäo. RESULTADOS: nos três métodos observou-se diferença significativa no escore embrionário entre o grupo de grávidas (n=53) e näo grávidas (n=84) (p<0,0001). No escore 1, avaliando-se apenas o número de células, observou-se maior taxa de gravidez (70 por cento) e taxa de implantaçäo (42 por cento) nas transferências de embriöes com média dos blastômeros acima de 8. O escore 2, baseado num escore total de quatro pontos (clivagem, número de blastômeros, fragmentaçäo e simetria), mostrou aumento nas taxas de gravidez (52,8 por cento) e implantaçäo (31 por cento) nos escores acima de 2. No escore 3, baseado no número de células e no grau morfológico, também as taxas de gravidez e de implantaçäo elevaram-se de acordo com o aumento do escore médio dos embriöes transferidos. CONCLUSÄO: Os três sistemas de escore avaliados em embriöes de 3º dia, correlacionaram-se positivamente com taxa de gravidez e taxa de implantaçäo

La investigacion cientifica con celulas embrionales humanas / Medicine research with human embryo stem cells

El uso de células madre (stem cells) en, medicina tiene actualmente una importancia notable, sobre todo en el ámbito de los tumores hemáticos, la cual está llamada a aumentar en los próximos años. Son muchas las patologías que en estos momentos no tienen una terapia eficaz (diabetes, Alzheimer, Parkinson...), y que podrían beneficiarse del uso de este tipo de células. La investigación en este campo no puede tener más que una valoración positiva desde el punto de vista ético, siempre que respete la vida humana desde su comienzo. Este artículo intenta mostrar cómo la extracción de células madre del embrión humano, con su consiguiente destrucción, no es un camino adecuado para esta investigación, y que se puede acudir a otras fuentes celulares que no presentan ningún inconveniente ético.

Sera from chronic chagasic patients depress cardiac electrogenesis and conduction

We report results obtained with sera from 58 chronic chagasic patients that were evaluated for effects on heart rate and atrioventricular (AV) conduction in isolated rabbit hearts and screened for the presence of muscarinic and beta-adrenergic activity. We show that sera from 26 patients decreased heart rate, while 10 increased it and 22 had no effect. Additionally, sera from 20 of the 58 patients blocked AV conduction. Muscarinic activation seems to be involved in both effects, but is not the only mechanism, since atropine did not antagonize the decrease in heart rate in 23 percent of sera or AV block in 40 percent. Sera from patients with complex arrhythmias were significantly more effective in depressing both heart rate and AV conduction. Sera that induce increases in heart rate seem to operate exclusively through beta-adrenergic activation. Two of these sera, evaluated with respect to intercellular communication in primary cultures of embryonic cardiomyocytes were able to block gap junction conductance evaluated by a dye injection technique after 24-h exposure. The mechanisms underlying this uncoupling effect are currently being investigated

Relaçäo entre qualidade do embriäo e índice de gravidez após fertilizaçäo "in vidro" e transferência de embriäo / Relation between quality of embryo and pregnancy rate after in vitro fertilization and embryo transfer

Um importante fator que influencia o índice de gravidez após a fertilizaçäo "in vitro" e tranferência de embriäo (FIVETE) é a qualidade morfológica dos embriöes transferidos para o útero. Este trabalho mostra esta relaçäo em 59 transferências. As pacientes foram separadas em 4 grupos. Grupo A: nenhum dos embriöes transferidos era de boa qualidade, näo ocorrendo gravidez neste grupo; Grupo B: dos embriöes transferidos apenas um era de boa qualidade, ocorrendo 20 por cento de gravidez; Grupo C: dos embriöes dois eram de boa qualidade, ocorrendo 27,27 por cento de gravidez; Grupo D: dos embriöes transferidos, pelo menos três eram de boa qualidade, ocorrendo 37,50 por cento de gravidez neste grupo. Näo houve diferença significante na ocorrência de gravidez entre os grupos B, C e D. Porém, quando comparados com o grupo A, a diferença foi estatisticamente significante, p>0,05.