Reprogramming of Human Somatic Cells Using Human and Animal Oocytes
Reprogramming of Human Somatic Cells Using Human and Animal Oocytes
复制标题
DOI：
10.1089/clo.2009.0004
复制
发表时间：
2009-06-01
期刊：
影响因子：
--
通讯作者：
Lanza, Robert
中科院分区：
文献类型：
--
作者：
Chung, Young;Bishop, Colin E.;Lanza, Robert
利用动物卵母细胞对人类体细胞进行重编程的研究重新引起人们的兴趣。在这里，我们比较了使用从动物和人类来源获得的卵母细胞的人类体细胞核的重编程。采用单胚转录组扩增和全局基因表达分析对桑椹胚期胚胎的基因表达进行了比较分析。基因组DNA指纹图谱和PCR分析证实，克隆胚胎的核基因组来源于供体体细胞。尽管人-人、人-牛和人-兔克隆在形态上相似，并以大致相同的速率（分别为39%、36%和36%）继续发育到桑椹胚阶段，但供体基因组的重编程模式却截然不同。与种间克隆相反，人-人胚胎的基因表达谱显示，供体细胞核通过广泛的上调进行了广泛的重编程，并且表达模式在正常对照胚胎中的关键上调中相似。为了解释母体基因表达，从相应的桑椹胚阶段胚胎谱中减去去核卵母细胞转录组谱。t检验比较（中值归一化数据@ fc > 4; p < 0.005）在人体外受精（IVF）胚胎和人-牛或人-兔种间体细胞移植（iSCNT）胚胎之间发现2400至2950个基因差异表达，其中大多数（60-70%）下调，而牛和兔卵母细胞谱之间的相同比较发现根本没有差异。与iSCNT胚胎相反，与年龄匹配的IVF胚胎相比，人-人克隆的表达谱显示，几乎所有差异表达的基因在克隆中均上调。重要的是，人类卵母细胞显著上调Oct-4，Sox-2和nanog（分别为22倍，6倍和12倍），而牛和兔卵母细胞没有显示出差异或下调这些关键的多能性相关基因，有效地沉默它们。如果没有适当的重编程，这些数据对这些不一致的动物卵母细胞来源产生患者特异性干细胞的潜在用途提出了质疑。
There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc > 4; p < 0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.