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

项目摘要 衰老具有复杂的生物学基础，其特征是细胞和生理功能的逐渐丧失 并且这种恶化与退行性疾病密切相关。在骨髓中， 降低造血干细胞（HSC）自我更新和分化成淋巴系的能力， 导致免疫功能受阻和对多个组织的系统性影响，例如损伤后的肌肉修复。 来自年轻受者的骨髓已被证明可以使衰老的骨髓恢复活力，以及全身性地恢复活力。 其他组织。然而，确切的HSC和祖细胞状态以及其他驱动HSC分化的因素， 恢复活力的效果还没有被很好地理解。衰老过程中HSC和其他骨髓细胞类型的概况 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。基于TF的干预措施，如部分重编程，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， 预先确定的TF，通常是Yamanaka因子Oct 3/4、Sox 2、Klf 4和c-Myc，并且仅被 在一组选定的组织中得到证实。此外，HSC群体的多样性，包括 衰老细胞和长期更新状态（LT-HSC）等亚型，使主 监管机构的挑战，现有的方法不解决这种异质性。我们假设高- 来自不同年龄小鼠的HSC的分辨率单细胞RNA测序（scRNA-seq）将揭示推定的TF 这些候选者可以将老化的HSC重编程为LT-HSC， 静止状态能够恢复生态位，以逆转与年龄相关的表型。我们会分析分子 在单细胞分辨率的HSC中的老化特征，并使用这些数据来开发老化的度量， 提名TF以促进LT-HSC恢复和再生。我们将综合选定的TF， 筛选允许在体外和体内快速评价它们的重编程作用。将这些合并的 scRNA-seq读数的体内扰动将允许通过我们的老化评估HSC的再生。 淋巴/骨髓偏斜的特征和测量。表现出最强潜力的候选TF 将单独测试LT-HSC的复壮，并组合测试整个生物体的修饰 表型，包括增加的再增殖潜力，减少炎症因子，和改善 肌肉损伤后的修复调节HSC再生的新型TF的再利用作为新的 衰老相关疾病的治疗为细胞工程提供了新的框架。这项提议， 将转录组读出和筛选结合起来，以发现细胞状态的新调节因子， 在老龄化和更广泛的人类健康方面，TF为疾病干预奠定了基础。

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.