Spermatogonial Stem Cell Maintenance

精原干细胞维护

• 批准号: 10612942
• 负责人: ROBERT E BRAUN
• 金额: $ 40.28万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-10 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612942
• 关键词: Ablation; Adult; Alleles; Automobile Driving; Biological Assay; Busulfan; Cancer Etiology; Cancer Patient; Cell Cycle; Cell Cycle Regulation; Cell Fate Control; Cell Maintenance; Cell Nucleus; Cell Therapy; Cells; Chromatin; Chromatin Structure; Complement; Data; Detection; Development; Equilibrium; Frequencies; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Enhancement; Genetic Suppression; Germ Cells; Heterogeneity; Individual; Infertility; Knowledge; Male Contraceptive Agents; Male Infertility; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Normal Cell; Phenotype; Phosphorylation; Population; Production; Public Health; Publishing; Regenerative Medicine; Reserve Cell; Role; S phase; Sorting; Spermatogenesis; Spermatogonia; Supporting Cell; Testis; Tissues; Transposase; Work; ZNF145 gene; adult stem cell; age related; cdc Genes; exhaustion; fertility preservation; male; mutant; progenitor; reproductive; self-renewal; single nucleus RNA-sequencing; single-cell RNA sequencing; stem cell biomarkers; stem cell self renewal; stem cells; tissue repair; transcription factor

PROJECT SUMMARY/ABSTRACT Most adult tissues are maintained by resident adult stem cells that maintain the function and integrity of the tissue. As old cells die or are damaged, new cells are produced from adult stem cells. In many tissues there is emerging data suggesting the presence of both rapid-cycling and quiescent stem cells. Rapid cycling cells are actively engaged in tissue repair while slow-cycling, or G0-arrested cells, are reserve cells. Recently published work, and preliminary studies in this proposal, support the hypothesis that spermatogonial stem cells (SSCs) in the testis are heterogeneous in their cycling status and that disruption of the normal cell cycle can interfere with both self-renewal and differentiation. We have identified a subpopulation of spermatogonia that express EOMES, a T box transcription factor. Using lineage tracing we have shown that they contribute to steady-state spermatogenesis and to regeneration following germ cell ablation by busulfan. In Plzf mutant mice, which show an age-dependent depletion of SSCs, EOMES+ cells cycle more rapidly, suggesting that age-dependent depletion of SSCs is caused by proliferative exhaustion. The central hypothesis of this proposal is that spermatogonial stem cells (SSCs) are also heterogeneous with respect to their cycling status and that there are both rapid cycling and slow cycling SSCs. We propose that proper regulation of the cell cycle is critical for maintaining the homeostatic balance between self-renewal and differentiation and that loss of cell cycle regulation can lead to age-dependent loss of SSCs due to the mis-regulation of critical self-renewal genes. In Specific Aim 1 we will quantify the frequency of cell cycle asynchrony, the concordance of SSC marker expression and cycling status, and the effect of mutation of Plzf on both of these parameters. In Specific Aim 2 we will utilize an allele of Ki67 (Mki67-RFP) to flow sort cycling and non-cycling cells from the larger pool of GFRA1+ population. Single cell RNA sequencing (scRNAseq), and single nuclei Assay for Transposase Accessible Chromatin (snATACseq) of the cycling and non-cycling cells will provide and independent assessment of the non-cycling G0 population of SSCs, whether Eomes and other markers of SSCs cells are enriched within the population, and whether there is a previously unidentified population of G0 cells within the SSC pool. Lastly, In Specific Aim 3 we will assess how mutation of Batf, a regulator of Eomes expression, enhances the germ cell loss phenotype in Plzf lu/lu mutants, and assess the role of the mis-regulation of cell cycle genes in the driving the hyperproliferative phenotype.

项目摘要/摘要 大多数成年组织由居民成年干细胞维持，以维持的功能和完整性 组织。由于旧细胞死亡或损坏，新细胞是由成年干细胞产生的。在许多组织中 新兴数据表明存在快速偶联和静止的干细胞。快速循环细胞是 储备金是储备细胞，在慢速循环或G0放置的细胞时积极参与组织修复。最近出版 该提案中的工作和初步研究支持了精子干细胞（SSC）中的假设 睾丸的循环状态是异质的，正常细胞周期的破坏会干扰 自我更新和差异化。我们已经确定了表达Eomes的精子的亚群， T盒转录因子。使用谱系跟踪我们已经证明它们有助于稳态 精子发生和再生在Busulfan生殖细胞消融后。在PLZF突变小鼠中，显示 EOMES+细胞循环依赖于年龄的SSC耗尽，表明年龄依赖性 SSC的耗竭是由增生性疲惫引起的。该提议的核心假设是 精子干细胞（SSC）在其循环状态方面也是异质的，并且存在 快速循环和缓慢的骑自行车SSC。我们建议对细胞周期的适当调节对于 保持自我更新和分化之间的体内平衡以及细胞周期的丧失 由于关键自我更新基因的错误调节，调节可能导致SSC的年龄依赖性丧失。在 特定目标1我们将量化细胞周期异步的频率，SSC标记的一致性 表达和循环状态，以及PLZF突变对这两个参数的影响。在特定目标2中 我们将利用Ki67（MKI67-RFP）的等位基因从较大的池中流动循环和非循环细胞 GFRA1+人口。单细胞RNA测序（SCRNASEQ）和转座酶的单核测定法 循环和非循环细胞的可访问的染色质（SNATACSEQ）将提供并独立 评估SSC的非循环G0人群，SSC细胞的EOME和其他标记是否为 在人群中富集，以及以前未确定的G0细胞中的人群 SSC池。最后，在特定目标3中，我们将评估eomes表达的调节剂BATF突变，如何 增强PLZF LU/LU突变体中的生殖细胞损失表型，并评估细胞周期错误调节的作用 驱动高增殖表型的基因。