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Derivation of Primordial Germ Cells from Human Embryonic and Induced Pluripotent Stem Cells Is Significantly Improved by Coculture with Human Fetal Gonadal Cells

机译:与人胎儿性腺细胞共培养显着改善了人胚和诱导多能干细胞中原始生殖细胞的衍生

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The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real-time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure. STEM CELLS 2009;27:783-795
机译:从人类胚胎干细胞(hESCs)或人类诱导的多能干(hIPS)细胞衍生生殖细胞代表了理想的实验模型和治疗不育症的潜在策略。在当前研究中,我们开发了一种三重生物标志物测定法,用于通过首先评估体内头三个月期间人PGC的形成来鉴定和分离人原始生殖细胞(PGC)。接下来,我们将该技术应用于表征多能细胞的体外衍生PGC(iPGC)。我们的结果表明,hESC在人胎儿性腺基质细胞上的共分化显着提高了产生iPGC的效率。此外,无论来自人胚泡(hESCs)的内部细胞团,还是人皮肤成纤维细胞(hIPS)的重新编程,各种多能细胞系之间的效率都相当。为了更好地表征iPGC,我们进行了实时聚合酶链反应,微阵列和亚硫酸氢盐测序。我们的结果表明,分化后第7天的iPGC在转录上不同于体细胞,表达与多能性和生殖细胞发育相关的基因,同时抑制与体细胞分化相关的基因(特别是多个HOX基因)。使用亚硫酸氢盐测序,我们显示iPGCs在分化的第7天从差异甲基化的印迹区域启动了印迹清除。但是,源自hIPS细胞的iPGC不能有效地启动印迹清除。总之,我们的结果表明,源自在hFGS细胞上分化的多能细胞的三重阳性iPGC对应于已启动的早孕生殖细胞(在9周之前),这些细胞已启动了印迹消除过程。干细胞2009; 27:783-795

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    《STEM CELLS》 |2009年第4期|783-795|共13页
  • 作者单位

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles;

    Department of Medicine, University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles;

    Department of Pathology and Laboratory Medicine, University of California, Los Angeles|David Geffen School of Medicine, University of California, Los Angeles|Jonsson Comprehensive Cancer Center, University of California, Los Angeles|Molecular Biology Institute, University of California, Los Angeles|Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles|Jonsson Comprehensive Cancer Center, University of California, Los Angeles|Molecular Biology Institute, University of California, Los Angeles|Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles|Jonsson Comprehensive Cancer Center, University of California, Los Angeles|Molecular Biology Institute, University of California, Los Angeles|Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and University of California, Los Angeles;

    David Geffen School of Medicine, University of California, Los Angeles|Jonsson Comprehensive Cancer Center, University of California, Los Angeles|Molecular Biology Institute, University of California, Los Angeles|Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and University of California, Los Angeles|Department of Biological Chemistry, University of California, Los Angeles;

    Department of Molecular Cell and Developmental Biology, University of California, Los Angeles|College of Letters and Science, University of California, Los Angeles|Jonsson Comprehensive Cancer Center, University of California, Los Angeles|Molecular Biology Institute, University of California, Los Angeles|Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and University of California, Los Angeles;

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