Differentiating embryonic stem cells into developing germ line

描述（由申请人提供）：在美国，大约每10对育龄夫妇中就有1对被诊断患有不孕症。不孕症不会因种族、性别或民族而产生偏见。人类不育的根本原因往往是未知的。然而，异常的形成，或对负责创造卵子和精子的谱系的不可逆转的损伤可能导致成年后的不育。我们认为，了解人类卵子和精子分化（也称为生殖系分化）机制的最佳模型之一涉及生殖系分化。 从体外多能干细胞中分离细胞。目前，生殖系体外分化领域受到以下因素的阻碍：缺乏胚胎中人类生殖系发育的知识、体外分化后的低产量、生殖系分化潜能在品系之间的未知变异性以及未能使用生殖系异种移植模型作为功能测定。为了克服这些瓶颈，我们建议使用RNA测序和全基因组亚硫酸氢盐测序来开发妊娠期间体内人类生殖系细胞的全面转录组和DNA甲基化组图谱。这将用于体外获得的生殖系细胞的转录和表观遗传分期。接下来，我们建议通过使用基因组编辑技术将基因整合到安全港位点中，在一组9个充分表征的hESC系中诱导PRDM14，NANOS3和DAZL的异位表达。我们将使用下一代单细胞基因表达面板测量生殖系身份，并在早期人类生殖系发育中稳定去甲基化的基因座处进行去甲基化。最后，我们建议使用生殖系异种移植试验，在体外雄性生殖系细胞移植到化疗导致不育的小鼠睾丸。该试验的终点将涉及成熟生殖系标志物的集落形成、增殖和表达。将异种移植结果与来自妊娠期睾丸的人精原细胞和人生殖系祖细胞的对照异种移植进行比较。该项目的结果将对体外生殖系分化领域产生长期持久的影响，因为它提供了多个良好表征的hESC系的分化效率的测量，以及最先进的基因编辑技术和功能测定的结合，以改善和确认体外生殖系的身份。

DESCRIPTION (provided by applicant): Approximately 1 in 10 couples of reproductive age in America are diagnosed with infertility. Infertility does not bias by race, gender or ethnicity. Underlying causes of human infertility are often unknown. However, abnormal formation, or irreversible damage to the lineage responsible for creating egg and sperm can cause infertility as an adult. We propose that one of the best models to understand mechanism of human egg and sperm differentiation (also known as germline differentiation) involve differentiating germline cells from pluripotent stem cells in vitro. Currently, the field of germline differentiation in vito is hindered by a lack of knowledge of human germline development in the embryo, low yield following in vitro differentiation, unknown variability in germline differentiation potential betwen lines, and failure to use the germline xenotransplantation model as a functional assay. To overcome these bottlenecks we propose to develop a comprehensive transcriptome and DNA methylome map of in vivo human germline cells during gestation using RNA- sequencing and whole genome bisulfite-sequencing. This will be used to transcriptionally and epigenetically stage germline cells acquired in vitro. Next, we propose to induce ectopic expression of PRDM14, NANOS3 and DAZL in a panel of nine well-characterized hESC lines by incorporating the genes into a safe harbor locus using genome-editing technology. We will measure germline identity using a next generation single-cell gene expression panel, and demethylation at loci that stably demethylate in early human germline development. Finally, we propose to use the germline xenotransplantation assay to transplant in vitro male germline cells into the testes of mice rendered infertile by chemotherapy. The endpoint of this assay will involve colony formation, proliferation and expression of mature germline markers. Xenotransplantation outcomes will be compared to control xenotransplantation of human spermatogonia and progenitor human germline cells from gestational stage testes. Results from this project will have a long lasting impact on the field of germline differentiation in vitro as it provides a measure of differentiation efficiency across multiple well characterized hESC lines, as well as incorporation of state-of-the art gene editing technology and functional assays to improve and confirm germline identity in vitro.