Discovery and manipulation of transcription factors to restore long term stem cell repopulation in aged bone-marrow

项目摘要 衰老具有复杂的潜在生物学特征，其特征是细胞和生理功能的进行性丧失 这种恶化与退行性疾病密切相关。在骨髓中，显著老化 降低造血干细胞(HSCs)自我更新和分化为淋巴系的能力， 导致免疫功能障碍和对多个组织的全身影响，如肌肉损伤后的修复。 来自年轻受者的骨髓被证明可以使衰老的骨髓恢复活力，而且在系统地 其他纸巾。然而，确切的HSC和祖细胞状态以及其他推动 恢复活力的效果还没有被很好地理解。造血干细胞和其他骨髓细胞类型在衰老过程中的分布 并了解控制HSC自我更新及其活性变化的转录因子(TF) 可以提供新的治疗方法，有可能逆转血液特异性和 衰老对整个动物的影响。以信托基金为基础的干预措施，如部分重新编程，已显示出希望 促进干细胞循环和再生。然而，目前的方法仅限于一小部分 预定的TF，通常是山中因数Oct3/4，Sox2，Klf4和c-Myc，并且只被 在一组精选的组织中展示。此外，HSC种群的多样性，包含两者 衰老细胞和长期更新状态(LT-HSC)等亚型，发现了MASTER 监管机构提出了挑战，而现有的方法并没有解决这种异质性。我们假设很高- 来自不同年龄的小鼠肝干细胞的单细胞RNA测序(scRNA-seq)将揭示假定的转铁蛋白 衰老过程的调节者，这些候选人可以将老化的HSC重新编程为LT-HSC和 能够恢复生态位以逆转与年龄相关的表型的静止状态。我们将分析分子 在单个细胞分辨率下的HSC中的老化特征，并使用这些数据来开发老化和 提名TF以促进LT-HSC的恢复和年轻化。我们将合成精选的TF用于池化 筛选允许快速评估它们在体外和体内的重编程效果。将这些池连接起来 体内scRNA-seq读数的扰动将允许通过我们的衰老来评估HSC的年轻化 淋巴/髓系歪斜的征象和测量。表现出最大潜力的候选人 对于LT-HSCs的返老还童，将进行单独和联合测试以进行整体生物体的修饰 表型，包括增加再种群潜力，减少炎症因子，以及改善 肌肉修复对损伤的反应。调节HSC返老还童的新型转录因子的再利用 衰老相关疾病的治疗为细胞工程提供了一个新的框架。这项提议， 结合转录读数和筛选发现新的细胞状态调节因子，作为 为以工作组为基础的疾病干预奠定基础，包括在老龄化和更广泛的人类健康方面。

Project Summary Aging has a complex underlying biology characterized by a progressive loss of cellular and physiological function and this deterioration is strongly correlated with degenerative disease. In the bone marrow, aging markedly reduces the capacity of hematopoietic stem cells (HSCs) to self-renew and differentiate into lymphoid lineages, resulting in hindered immune function and systemic effects on multiple tissues, such as muscle repair after injury. Bone marrow from young recipients has been shown to rejuvenate aged bone marrow as well as systemically in other tissues. However, the exact HSC and progenitor cell states as well as other factors that drive the rejuvenating effects are not well understood. Profiling of HSCs and other bone marrow cell types during aging and an understanding of the transcription factors (TFs)-that control HSC self-renewal and their changes in activity during aging could provide new therapeutic approaches with the potential to reverse both blood-specific and whole-animal effects of aging. TF-based interventions, such as partial reprogramming, have shown promise to promote stem-cell cycling and regeneration. However, current approaches are limited to a small set of predetermined TFs, commonly the Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc, and have only been demonstrated in a select set of tissues. Furthermore, the diversity of the HSC population, containing both senescent cells and long-term renewing state (LT-HSC) among other subtypes, makes discovery of master regulators challenging, and existing approaches do not address this heterogeneity. We hypothesize that high- resolution single cell RNA sequencing (scRNA-seq) of HSCs from mice of different ages will reveal putative TF regulators of the aging process, and that these candidates can reprogram aged HSCs towards LT-HSC and quiescent states capable of niche restoration to reverse age-associated phenotypes. We will profile molecular signatures of aging in HSCs at single cell resolution and use these data to both develop metrics for aging and nominate TFs to promote LT-HSC restoration and rejuvenation. We will synthesize selected TFs for pooled screening allowing for rapid evaluation of their reprogramming effects in vitro and in vivo. Coupling these pooled perturbations in vivo with scRNA-seq readouts will allow for evaluation of HSC rejuvenation via our aging signatures and measurement of lymphoid/myeloid skew. Candidate TFs that demonstrate the strongest potential for rejuvenation of LT-HSCs will be tested individually and in combination for modification of whole-organism phenotypes, including increased repopulation potential, reduction of inflammatory factors, and improvement of muscle repair in response to injury. The repurposing of novel TFs regulating HSC rejuvenation as new therapeutics for aging-associated disease provides a new framework for cellular engineering. This proposal, coupling transcriptomic readouts and screening for discovery of new regulators of cell states, serves as the foundation for TF-based interventions for disease, both in aging and in broader human health.