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：
9174849
负责人：
Amander Clark
金额：
$ 44.21万
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/9174849
关键词：
Adult Affect Age Alpha Cell 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 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 Pregnancy Premature Ovarian Failure Proliferating Research Resources Scientist Seminiferous tubule structure Specific qualifier value Spermatogonia Stem cells 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 pre-clinical progenitor public health relevance reproductive reproductive success research clinical testing sperm cell 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，大约十分之一的育龄夫妇被诊断患有不孕症。不孕不育不因种族、性别或民族而存在偏见。人类不孕不育的根本原因往往是未知的。然而，异常形成或对负责产生卵子和精子的谱系的不可逆转的损害可能会导致成年不孕。我们提出，了解人类卵子和精子分化（也称为种系分化）机制的最佳模型之一涉及种系分化来自体外多能干细胞的细胞。目前，体外种系分化领域受到以下因素的阻碍：缺乏对胚胎中人类种系发育的了解、体外分化后的产量低、系间种系分化潜力的未知变异性以及未能使用种系异种移植模型作为模型。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细胞的全面转录组和 DNA 甲基化组图谱。这将用于对体外获得的生殖细胞进行转录和表观遗传学分期。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基因座中，在一组九个充分表征的 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组来测量种系身份，以及在早期人类种系发育中稳定去甲基化的位点的去甲基化。最后，我们建议使用种系异种移植试验将体外雄性种系细胞移植到因化疗而导致不育的小鼠的睾丸中。该测定的终点将涉及集落形成、增殖和成熟种系标记的表达。异种移植结果将与来自妊娠期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种系分化领域产生长期持续的影响，因为它提供了跨多个特征明确的 hESC 系的分化效率的衡量标准，并结合了最先进的基因编辑技术和功能测定改进和确认体外种系同一性。