Histone Demethylase-Assisted Somatic Cell Nuclear Transfer Facilitates Derivation of Human Pluripotent Stem Cells

The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing derivation of NTESCs from all oocyte donors tested using adult AMD patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine. Overall design: Here we perform RNA-seq based transcriptome profiling in human Donor (fibroblast cells), in vitro fertilized embryos at 8-cell stages (IVF_8Cell), somatic cell nuclear transfer embryos at 8-cell stages (SCNT_8Cell), SCNT assisted by KDM4A 8-cell embryos (SCNT_KDM4A_8Cell). Besides, we also perform RNA-seq in Control human ES cells (CTR_hES) and SCNT assisted by KDM4A derived human ES cells (NTK) with duplicates.Â

利用体细胞核移植（somatic cell nuclear transfer, SCNT）技术获取人类胚胎干细胞（human embryonic stem cell, hESC）的效率极低，极大限制了其潜在应用价值。人类体细胞核移植胚胎的囊胚形成率偏低，且仅在部分卵母细胞供体中可获得。我们此前在小鼠研究中证实，通过异位表达H3K9me3去甲基化酶Kdm4d以降低组蛋白H3赖氨酸9三甲基化（histone H3 lysine 9 trimethylation, H3K9me3）水平，可显著改善体细胞核移植胚胎的发育状况。本研究发现，过表达另一类相关的H3K9me3去甲基化酶KDM4A可优化人类体细胞核移植流程；且与小鼠研究结果一致，人类体细胞基因组中的H3K9me3是体细胞核移植重编程的障碍。KDM4A过表达可通过促进转录重编程，显著提升人类体细胞核移植胚胎的囊胚形成率，使得研究人员可从所有受试卵母细胞供体中，利用成年AMD患者的体细胞核成功获取核移植胚胎干细胞（nuclear transfer embryonic stem cell, NTESC）。这一保守的机制性研究发现，在包括再生医学在内的多个领域中，均具备优化体细胞核移植技术的应用潜力。

整体实验设计：本研究对人类供体细胞（成纤维细胞）、8细胞期体外受精胚胎（IVF_8Cell）、8细胞期体细胞核移植胚胎（SCNT_8Cell）、经KDM4A辅助的8细胞期体细胞核移植胚胎（SCNT_KDM4A_8Cell）开展基于RNA测序的转录组分析。此外，我们还对对照组人类胚胎干细胞（CTR_hES）以及经KDM4A辅助构建的核移植胚胎干细胞（NTK）设置生物学重复并进行RNA测序。