Differentiating embryonic stem cells into developing germ line

描述(申请人提供)：在美国，大约十分之一的育龄夫妇被诊断为不孕不育。不孕不育不会因种族、性别或民族而产生偏见。人类不孕不育的根本原因通常是未知的。然而，异常形成或对负责产生卵子和精子的谱系造成不可逆转的损害，可能会导致成年后的不育。我们认为，理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到生殖系的分化。 在体外从多能干细胞中获得细胞。目前，由于对人类生殖系在胚胎中的发育缺乏了解，体外分化后的产量较低，种系之间的种系分化潜力未知差异，以及未能将生殖系异种移植模型用作功能性实验，阻碍了体外生殖系分化领域的发展。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盐测序来建立体内人类生殖系细胞在妊娠期间的全面转录组和DNA甲基组图谱。这将被用来从转录和表观遗传学的角度对体外获得的生殖细胞进行分期。接下来，我们建议利用基因组编辑技术，通过将PRDM14、NANOS3和DAZL基因整合到一个安全港基因座，在一组9个特征良好的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板来测量生殖系的同一性，并在人类生殖系发育的早期稳定地去甲基化的基因座上进行去甲基化。最后，我们建议使用生殖系异种移植试验，在体外将雄性生殖系细胞移植到因化疗而不育的小鼠的睾丸中。这项试验的终点将涉及克隆形成、增殖和成熟种系标记的表达。异种移植的结果将与来自妊娠阶段睾丸的人类精原细胞和人类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的影响，因为它提供了对多个特征良好的hESC系的分化效率的测量，以及结合最先进的基因编辑技术和功能分析来改进和确认体外生殖系的鉴定。

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.