New tools for cell reprogramming and conversion: Possible applications to livestock

Somatic cell nuclear transfer and iPS are both forms of radical cell reprogramming able to transform a fully differentiated cell type into a totipotent or pluripotent cell. Both processes, however, are hampered by low efficiency and, in the case of iPS, the application to livestock species is uncertain. Epigenetic manipulation has recently emerged as an efficient and robust alternative method for cell reprogramming. It is based upon the use of small molecules that are able to modify the levels of DNA methylation with 5-azacitidyne as one of the most widely used. Among a number of advantages, it includes the fact that it can be applied to domestic species including pig, dog and cat. Treated cells undergo a widespread demethylation which is followed by a renewed methylation pattern induced by specific chemical stimuli that lead to the desired phenotype. A detailed study of the mechanisms of epigenetic manipulation revealed that cell plasticity is achieved through the combined action of a reduced DNA methyl transferase activity with an active demethylation driven by the TET protein family. Surprisingly the same combination of molecular processes leads to the transformation of fibroblasts into iPS and regulate the epigenetic changes that take place during early development and, hence, during reprogramming following SCNT. Finally, it has recently emerged that mechanic stimuli in the form of a 3D cell rearrangement can significantly enhance the efficiency of epigenetic reprogramming as well as of maintenance of pluripotency. Interestingly these mechanic stimuli act on the same mechanisms both in epigenetic cell conversion with 5-Aza-CR and in iPS. We suggest that the balanced combination of epigenetic erasing, 3D cell rearrangement and chemical induction can go a long way to obtain ad hoc cell types that can fully exploit the current exiting development brought by gene editing and animal cloning in livestock production.

New tools for cell reprogramming and conversion: Possible applications to livestock

Somatic cell nuclear transfer and iPS are both forms of radical cell reprogramming able to transform a fully differentiated cell type into a totipotent or pluripotent cell. Both processes, however, are hampered by low efficiency and, in the case of iPS, the application to livestock species is uncertain. Epigenetic manipulation has recently emerged as an efficient and robust alternative method for cell reprogramming. It is based upon the use of small molecules that are able to modify the levels of DNA methylation with 5-azacitidyne as one of the most widely used. Among a number of advantages, it includes the fact that it can be applied to domestic species including pig, dog and cat. Treated cells undergo a widespread demethylation which is followed by a renewed methylation pattern induced by specific chemical stimuli that lead to the desired phenotype. A detailed study of the mechanisms of epigenetic manipulation revealed that cell plasticity is achieved through the combined action of a reduced DNA methyl transferase activity with an active demethylation driven by the TET protein family. Surprisingly the same combination of molecular processes leads to the transformation of fibroblasts into iPS and regulate the epigenetic changes that take place during early development and, hence, during reprogramming following SCNT. Finally, it has recently emerged that mechanic stimuli in the form of a 3D cell rearrangement can significantly enhance the efficiency of epigenetic reprogramming as well as of maintenance of pluripotency. Interestingly these mechanic stimuli act on the same mechanisms both in epigenetic cell conversion with 5-Aza-CR and in iPS. We suggest that the balanced combination of epigenetic erasing, 3D cell rearrangement and chemical induction can go a long way to obtain ad hoc cell types that can fully exploit the current exiting development brought by gene editing and animal cloning in livestock production.(AU)

Epigenética na reprogramação celular e desenvolvimento embrionário.

A epigenética tem se destacado como a principal moduladora das funções celulares e reguladora da expressão gênica, seja pela ativação ou repressão da atividade transcricional. Além disso, a epigenética está relacionada diretamente a processos reprodutivos como a reprogramação celular e o desenvolvimento embrionário. Em um primeiro estudo, foi avaliado o efeito do extrato de oócitos em vesícula germinativa (VG), isoladamente, ou em associação com o inibidor de deacetilase, Scriptaid, sobre o potencial de reprogramação celular em células somáticas suínas. Foi observada a formação de colônias semelhantes à células-tronco pluripotentes aproximadamente duas semanas após o tratamento com o extrato de oócitos ou extrato de oócitos associado ao Scriptaid. O número de colônias, no dia de aparecimento e 48 horas após esse período, foi semelhante entre as células tratadas somente com o extrato de oócitos ou em associação com Scriptaid. Foi observada ainda a ativação parcial de genes de pluripotência celular e de genes reguladores de modificações de cromatina e de metilação de DNA, como o Ezh2 e o Dnmt1, três dias após o tratamento com extrato de oócitos. No entanto, 15 dias após o tratamento, esses níveis retornaram aos níveis do controle. Esses dados sugerem que o extrato de oócitos em estádio de VG é capaz de induzir uma reprogramação parcial nos fibroblastos suínos, caracterizada pela indução parcial de pluripotência e modulação de modificadores epigenéticos. Em um segundo estudo, foram caracterizados o co-fator ativador e remodelador de cromatina (BRG1), e a lisina demetilase 1 A (KDM1A), durante o desenvolvimento embrionário de suínos. A KDM1A atua na desmetilação da mono e dimetilação da lisina 4 da histona 3 (H3K4m3 e H3K4me2, respectivamente). Primeiramente, foi verificado que as proteínas desses fatores não estão presentes no núcleo de oócitos no estádio de metáfase II, porém estão presentes no núcleo da maioria dos embriões durante os dias 3-4 do desenvolvimento embrionário, o que coincide com o momento da ativação do genoma embrionário (EGA), na espécie suína. Além disso, utilizando um modelo de alta e baixa competência para o desenvolvimento, foi verificado que a expressão de RNAm desses fatores são regulados durante o desenvolvimento embrionário e estão correlacionados com a expressão de RNAm de outras enzimas demetilases de lisinas e com níveis de metilação da H3K4me e H3K4me2 durante a EGA. Observou-se ainda que os níveis proteicos dos fatores BRG1 e KDM1A parecem ter relação com o numero de células por embrião durante a EGA. Esses dados sugerem que esses fatores podem apresentar um envolvimento na regulação da H3K4 durante a ativação do genoma e consequente no desenvolvimento embrionário.

Epigenetic programming is the main mechanism regulating cell function and gene expression, through the activation or repression of transcriptional activity. In addition, epigenetics is closely related to reproductive events such as cell reprogramming and embryo development. In the first study, the effects of the germinal vesicle (GV) oocyte extract alone, or in combination with the deacetylase inhibitor Scriptaid, on porcine somatic cell reprogramming were evaluated. The formation of stem cell-like colonies were observed approximately two weeks after treatment with oocyte extract or oocyte extract plus Scriptaid. The colony number, at the time of appearance and after 48 hours, was similar between treatments. Partial activation of pluripotent, chromatin modifying and DNA methylating genes such as Ezh2 and Dnmt1, was observed three days after the oocyte extract treatment. However, the mRNA expression levels of the previous genes were similar to the control 15 days after treatment. This data suggest that GV oocyte extract is able to induce limited reprogramming in porcine fibroblasts, seen here by the partial activation of these genes. In the second study, brahmarelated gene-1 (BRG1), a cofactor and activator of chromatin modifications, and lysine demethylase 1A (Kdm1A), a repressor of gene expression, were characterized during porcine embryo development. Kdm1A is involved in the demethylation of both mono- and dimethylations, H3K4me and H3K4me2, respectively, on lysine 4 of histone 3. Firstly, we observed that proteins for both factors (BRG1 and Kdm1A) were absent in the nuclei of metaphase II oocytes, however, the proportion of nuclear localization increased on day 3-4 of embryo development. This time point coincides with the embryonic genome activation (EGA) in swine. Furthermore, using a well-established model of embryo developmental competence, based on time of first cleavage, it was verified that these factors were regulated during embryo development and are correlated with mRNA expression of other demethylases and H3K4me and H3K4me2 levels during EGA. It was also observed that BRG1 and Kdm1A levels are correlated with embryo cell numbers during EGA. These data suggest that BRG1 and Kdm1A participate in the regulation of H3K4 methylation during embryonic genome activation, and consequently, embryo development in swine.

Modelagem neuronal de pacientes com distrofia muscular de Duchenne utilizando células pluripotentes induzidas

A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença.

The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient's life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease.