An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

猪因其在解剖学、生理学与遗传学层面与人类存在高度相似性，成为研究人类遗传疾病的优质模型动物。此前虽已有研究通过转基因体细胞与体细胞核移植（somatic cell nuclear transfer, SCNT）技术培育出转基因猪，但其克隆效率极低。本研究报道了一种改良方法，可从猪诱导多能干细胞（porcine induced pluripotent stem cells, piPSCs）制备二倍体克隆胚胎：采用阿非迪霉素与诺考达唑双重阻断法，将piPSCs同步化至G2/M期。我们首先验证了该同步化方法对本研究中piPSC细胞系的作用效果，随后对传统体细胞核移植方案进行优化以筛选可行流程。尤为关键的是，移除激活后6-二甲基氨基嘌呤（6DMAP）处理步骤，可显著提升假第二极体（pseudo-second-polar bodies, p2PB）的排出率（以激活后4小时峰值计，81.3% vs 15.8%）。此外，即时激活方案相较于延迟激活方案，可获得更多囊胚（以融合胚胎数为统计基数，31.3% vs 16.0%）。免疫荧光实验结果证实了移除6DMAP处理步骤的效果，在一系列核移植操作中均观察到显著的p2PB排出现象。采用本改良方案，由中期piPSCs构建的重构胚胎在2-细胞期、4-细胞期及囊胚期均呈现正常形态，且正常核型比率较高。本研究建立了一种高效的新方法，可从同步化至细胞周期G2/M期的piPSCs制备具有活性的克隆胚胎，有望为更高效地利用piPSCs培育克隆猪提供新的可行途径。