Differentiating embryonic stem cells into developing germ line

将胚胎干细胞分化为发育中的种系

基本信息

批准号：
9384658
负责人：
Amander Clark
金额：
$ 43.22万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-20 至 2019-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9384658
关键词：
Adult Affect Age Americas Biological Biological Assay Cell Differentiation process Cell Lineage Cell Therapy Cell Transplants Cells Child Clinical Couples DNA Data Development Diagnosis Disease Ectopic Expression Embryo End Point Assay Environmental Hazards Epigenetic Process Ethnic Origin Event Exposure to Failure Fertility Future Gender Gene Expression Generations Genes Genetic Transcription Germ Cells Germ Lines Human Human Resources In Vitro Individual Infertility Injury Knowledge Logic Macaca mulatta Male Infertility Maps Measures Military Personnel Modeling Molecular Mus Outcome Parents Pluripotent Stem Cells Pre-Clinical Model Preclinical Testing Pregnancy Premature Ovarian Failure Proliferating Research Resources Scientist Seminiferous tubule structure Specific qualifier value Spermatogonia Technology Testing Testis Transplantation Woman Work Xenograft procedure aging population base bisulfite sequencing chemotherapy demethylation egg embryo/fetus embryonic stem cell genome editing human embryonic stem cell human embryonic stem cell line human pluripotent stem cell improved in vivo interest male men methylome next generation novel therapeutics occupational hazard progenitor public health relevance reproductive reproductive success sperm cell stem cell differentiation tool transcriptome transcriptome sequencing whole genome young cancer survivor

项目摘要

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.

描述（由申请人提供）：在美国，大约有十分之一的生殖年龄夫妇被诊断出患有不育。不孕症不会因种族，性别或种族而偏见。人类不孕症的根本原因通常是未知的。但是，导致产生鸡蛋和精子的谱系的异常形成或不可逆的损害可能导致成年人的不育。我们建议了解人卵和精子分化机制的最佳模型之一（也称为种系分化）涉及分化种系来自多能干细胞的细胞体外。当前，VITO的种系分化领域受到了对胚胎中人类种系发育的知识的阻碍，体外分化后的低产量，在生殖线差异潜在的细线中未知的变异性以及未能将种系异种移植模型作为功能分析。为了克服这些瓶颈，我们建议在妊娠期间使用RNA测序和整个基因组Bisulfite-sequesting开发体内人类种系细胞的全面转录组和DNA甲基甲基化图。这将用于在体外获得的转录和表观遗传学上的种系细胞。接下来，我们建议在使用基因组编辑技术中将基因纳入九个良好的hESC线的面板中诱导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种系认同，并在基因座的脱甲基化，该基因座在早期人类种系发育中稳定地脱甲基。最后，我们建议使用种系异种移植测定法将体外男性生殖细胞移植到化学疗法不育的小鼠的睾丸中。该测定的终点将涉及成熟种系标记的菌落形成，增殖和表达。异种移植结果将与妊娠期睾丸中人类精子和祖细胞生殖细胞的对照异种移植进行比较。该项目的结果将对体外种系分化领域产生持久的影响，因为它提供了多个良好特征hESC线的差异化效率，并纳入了最先进的基因编辑技术和功能分析，以改善并确认体外的生殖线身份。