Derivation of embryonic stem cells from cloned blastocysts using improved somatic cell nuclear transfer in common marmoset

The common marmoset (Callithrix jacchus) is a genetically modifiable non-human primate increasingly used in biomedical research. However, methods to preserve or expand valuable genetic resources in this species remain underdeveloped. Here, we established a method for deriving embryonic stem cells (ESCs) from blastocysts generated by somatic cell nuclear transfer (SCNT) in the marmoset. Injection of mRNA encoding the histone demethylase Kdm4d enabled efficient reprogramming of somatic nuclei, allowing blastocyst formation in 14.5% from fibroblasts. However, nuclear transfer ESCs (ntESCs) were not established from these blastocysts. Combining this method with a G9a histone methyltransferase inhibitor (RK-701) improved blastocyst quality and allowed derivation of ntESC lines, including wildtype and GFP-transgenic lines. These ntESCs exhibited normal karyotypes and pluripotency both in vitro and in vivo. Nuclear and mitochondrial DNA analyses confirmed their nuclear donor origin and cytoplasmic inheritance from recipient oocytes. Transcriptome analysis identified abnormally expressed genes in ntESCs present in a line-dependent and -independent manner, suggesting partial reprogramming resistance that may inform future optimization of SCNT in primates. Our study establishes marmoset SCNT method enabling derivation of ntESCs which may provide a new platform for preserving and engineering marmoset genetic resources. Comparative analysis of gene expression in ESCs derived from IVF, naturally fertilizaed (NAT), and SCNT in common marmoset.

普通狨猴（Callithrix jacchus）是一种可进行遗传修饰的非人灵长类动物，近年来在生物医学研究中的应用日益广泛。然而，针对该物种的珍贵遗传资源的保存与扩繁方法仍有待完善。本研究建立了一种从狨猴体细胞核移植（somatic cell nuclear transfer, SCNT）获得的囊胚中分离胚胎干细胞（embryonic stem cells, ESCs）的方法。向受体卵母细胞注射编码组蛋白去甲基化酶Kdm4d的mRNA，可实现体细胞核的高效重编程，使成纤维细胞来源的囊胚形成率达到14.5%。但未能从这些囊胚中建立核移植胚胎干细胞（ntESCs）。将该方法与G9a组蛋白甲基转移酶抑制剂（RK-701）联用后，囊胚质量得到显著改善，并成功建立了包括野生型及GFP转基因系在内的ntESC株系。这些ntESCs在体外及体内均表现出正常的核型与多能性。核DNA与线粒体DNA分析证实，其核基因组来源于供体细胞，细胞质组分则继承自受体卵母细胞。转录组分析发现，不同ntESC株系中存在株系依赖性及非依赖性的异常表达基因，提示存在部分重编程抵抗现象，这可为未来优化灵长类动物的体细胞核移植技术提供参考。本研究建立的狨猴体细胞核移植方法可实现ntESCs的分离，为狨猴遗传资源的保存与工程化改造提供了全新的研究平台。本研究还对普通狨猴体外受精（IVF）来源、自然受精（NAT）来源及体细胞核移植来源的胚胎干细胞的基因表达模式进行了比较分析。