Beneficial Effects of Reversine on In Vitro Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

Abstract

Abstract. Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei. As donor cells, we used miniature pig fetal fibroblasts transfected with a plasmid construct containing a mouse Oct-3/4 promoter and enhanced green fluorescent protein (EGFP) cDNA. When the nuclei of these transfected cells are reprogrammed to an undifferentiated state in the SCNT embryos, EGFP expression is expected to commence under the control of the Oct-3/4 promoter. After SCNT, the resulting embryos were treated with 5 μM reversine for different durations (0, 6, 12, 18 and 24 h) or at different concentrations (0, 1, 5 and 10 μM) of reversine for 12 h and then cultured in vitro. When embryos were treated with 5 μM reversine for 12 h, the blastocyst formation rate was significantly (P<0.01) higher than that of embryos without reversine treatment. However, the strength and pattern of EGFP expression in the embryos were not affected by the same treatment. A normal-looking fetus was obtained 21 days after transfer of embryos treated with 5 μM reversine for 12 h into recipients. The present findings indicate that treatment with reversine is beneficial for enhancement of the in vitro development of miniature pig SCNT embryos, although the underlying mechanism is still unclear. Key words: In vitro development, Miniature pig, Reprogramming, Reversine, Somatic cell nuclear transfer (J. Reprod. Dev. 56: [291][292][293][294][295][296] 2010) ecently, Chen et al. [1] reported that a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog could induce dedifferentiation of myogenic lineage-committed C2C12 cells to become multipotent mesenchymal progenitor cells in the concentration range of 1-10 μM. These progenitor cells could redifferentiate into osteoblasts or adipocytes under appropriate differentiation inducing conditions. The compound was named "reversine" because of its ability to reverse terminally differentiated cells to progenitor cells [1]. More recently, it was reported that human fibroblasts treated with reversine exhibited redifferentiation into skeletal muscle cells in vitro and in vivo [2]. Thus, reversine has the ability to reprogram somatic cells to a state of diverse plasticity that can be manipulated to differentiation into different cell types.Low cloning efficiency, which is often associated with low developmental rate of somatic cell nuclear transfer (SCNT) embryos, remains the major obstacle to use this technology in various fields of ani...

The synthetic purine reversine selectively induces cell death of cancer cells

“…, and even toward neuroectodermal lineage [Lee et al, 2009] and cardiomyocytes [Pikir et al, 2012]. Moreover, it was recently shown that the molecule increase mesenchymal stromal cells plasticity [Conforti et al, 2011] and can improve the efficiency of somatic cell nuclear transfer (SCNT) [Miyoshi et al, 2010]. Interestingly, in our initial study [Anastasia et al, 2006], we observed that reversine, besides inducing de-differentiation, greatly inhibited cell proliferation, leading to the formation of polyploid populations.…”

“…This led to the first screen for synthetic chemical agents that would turn on cell proliferation and induce myotube fission in differentiated C2C12 murine myocyte cultures, which culminated in the discovery of myoseverin [Rosania et al, 2000;Anastasia et al, 2006], and even toward neuroectodermal lineage [Lee et al, 2009] and cardiomyocytes [Pikir et al, 2012]. Moreover, it was recently shown that the molecule increase mesenchymal stromal cells plasticity [Conforti et al, 2011] and can improve the efficiency of somatic cell nuclear transfer (SCNT) [Miyoshi et al, 2010]. Interestingly, in our initial study [Anastasia et al, 2006], we observed that reversine, besides inducing de-differentiation, greatly inhibited cell proliferation, leading to the formation of polyploid populations.…”

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

“…Moreover, LatA treatment did not appear to have any detrimental effect on the morphology of early SCNT embryos. In porcine SCNT embryos, 10.4-35.5 μM (2.2-7.5 μg/ml) of CB has been used to inhibit extrusion of a pPB [21,[34][35][36]. However, LatA treatment effect like CB was provided by low density a little less than 20 times from 10 times in this study.…”

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei

“…Histone deacetylatase inhibitors (HDACi), such as Scriptaid, significantly increase the overall cloning efficiency through improvements in the histone acetylation on Histone 4 at lysine 8 (H4K8ac) in a pattern similar to that of the IVF embryos (Zhao et al 2010). Other HDACi, such as m-carboxycinnamic acid bishydroxamide (Song et al 2014), trichostatin A (Li et al 2008) and valproic acid (Kim et al 2011), have also been demonstrated to be beneficial to the in vitro development of porcine SCNT embryos (Miyoshi et al 2010, Zhao et al 2010, Samiec et al 2015.…”