Identification of Genes Important for Rejuvenation of Aged Hematopoietic Stem Cells

鉴定对老化造血干细胞再生重要的基因

• 批准号: 9764241
• 负责人: Hong Zhang
• 金额: $ 20.94万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764241
• 关键词: Affect; Age; Aging; Anemia; Biological Process; Blood Cells; C57BL/6 Mouse; CRISPR library; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell Aging; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Development; Disease; Elderly; Exhibits; GADD45A gene; GADD45G; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Guide RNA; Hematologic Neoplasms; Hematology; Hematopoietic stem cells; Homing; Immunity; Libraries; Lymphopoiesis; Mus; Myelogenous; Myeloproliferative disease; Natural regeneration; Pharmacology; Phenotype; Play; Population; Process; Production; Publishing; Regulation; Rejuvenation; Role; Site; Stem cells; System; Testing; Therapeutic; Transplantation; Validation; Youth; adult stem cell; age related; aged; aging gene; base; cell type; deep sequencing; genome editing; healthspan; hematopoietic differentiation; hematopoietic stem cell self-renewal; in vivo; insight; novel; nuclease; reconstitution; regenerative; self-renewal; stem cell differentiation; transcriptome

Abstract Hematopoietic stem cells (HSCs) are capable of extensive self-renewal and multi-lineage differentiation to maintain lifelong production of all blood cells. However, it is well documented that the self-renewal capacity of HSCs declines with age and old HSCs exhibit a skewed differentiation towards myeloid lineages at the expense of lymphopoiesis. These age-associated changes are responsible for decreased immunity and increased propensity for anemia and myeloid malignancies in the elderly. HSCs undergo complex changes in gene expression during aging. Whether these changes are responsible for HSC aging, and more importantly whether modulation of any of these changes can rejuvenate old HSCs is largely unknown and unexplored. We hypothesize that certain genes whose expression is up-regulated in HSCs during aging represent the “aging” factors and are responsible for the age-dependent decline in HSC self-renewal and function, whereas a class of down-regulated genes during HSC aging are the “fountain of youth” type of factors with their decreased expression leading to HSC aging. We further hypothesize that depletion of critical “aging” genes will rejuvenate old HSCs. To test these hypotheses, we will systematically identify genes that regulate HSC self-renewal and aging using an unbiased CRISPR-based in vivo genetic screen with the following two aims. In Aim 1, we will use a focused lentiviral CRISPR library to deliver the Cas9 nuclease and 21,268 single-guide RNAs (sgRNAs) to target 5,317 mouse genes that show altered expression in HSCs during aging. HSCs isolated from young mice will be infected with this CRISPR library and infected HSCs that carry different sgRNAs will be injected into stem cell-depleted recipient mice to repopulate competitively with each other in two rounds of transplantation. Altered HSC self-renewal and function will lead to either enrichment or depletion of HSCs. By analyzing the abundance of specific sgRNAs in HSCs and differentiated B, T or myeloid lineages using deep sequencing before and after transplantations, we will identify the corresponding genes that positively or negatively regulate HSC self- renewal and differentiation. In Aim 2, we will validate selected candidate genes identified in Aim 1 based on their significant enrichment or depletion in the screen and determine whether the processes affected by these candidate genes involve HSC homing, proliferation, quiescence or differentiation. Furthermore, we will test whether deletion in old HSCs of candidate “aging” genes and four genes (Gadd45a, Gadd45g, Runx1 and Selp) that have been shown to negatively regulate HSC self-renewal in young mice can enhance HSC self-renewal, reverse the skewed differentiation and thus rejuvenate old HSCs. The proposed studies will provide novel insight into the underlying mechanisms of HSC self-renewal, differentiation and aging. The translational potential of HSC rejuvenation discovered in the proposed studies will establish a paradigm for ameliorating age-associated diseases and promoting healthspan.

摘要 造血干细胞（HSC）能够广泛的自我更新和多谱系分化， 维持所有血细胞的终身生产。然而，有充分的证据表明，自我更新能力 的HSC随着年龄的增长而下降，并且老年HSC表现出向骨髓谱系的倾斜分化， 淋巴细胞生成的代价。这些与年龄相关的变化导致免疫力下降， 增加老年人贫血和骨髓恶性肿瘤的倾向。HSC经历复杂的变化 衰老过程中的基因表达。这些变化是否是HSC老化的原因，等等。 重要的是，这些变化中的任何一种的调节是否可以使老化的HSC再生在很大程度上是未知的， 未开发的我们假设某些基因在衰老过程中在HSC中表达上调， 代表“衰老”因素，并负责HSC自我更新的年龄依赖性下降， 功能，而在HSC衰老过程中下调的一类基因是“青春之泉”型， 这些因子的表达降低导致HSC老化。我们进一步假设， 关键的“老化”基因将使老化的HSC恢复活力。为了验证这些假设，我们将系统地识别 使用基于无偏CRISPR的体内遗传筛选调节HSC自我更新和衰老的基因 有以下两个目的。在目标1中，我们将使用聚焦慢病毒CRISPR库来递送Cas9 核酸酶和21，268个单向导RNA（sgRNA）靶向5，317个小鼠基因，这些基因显示出改变的 在衰老过程中HSC的表达。从年轻小鼠中分离的HSC将被这个CRISPR文库感染 将携带不同sgRNA的感染的HSC注射到干细胞耗尽的受体小鼠中， 在两轮移植中相互竞争地重新繁殖。HSC自我更新改变， 功能将导致HSC的富集或耗尽。通过分析特定sgRNA的丰度， 在HSC和分化的B、T或骨髓谱系中，使用深度测序前后， 移植，我们将确定相应的基因，积极或消极调节HSC自我， 更新和分化。在目标2中，我们将根据以下条件验证目标1中确定的选定候选基因： 它们在屏幕上的显着富集或耗尽，并确定是否受影响的过程 这些候选基因涉及HSC归巢、增殖、静止或分化。而且我们 将测试是否在老年HSC中缺失候选“衰老”基因和四个基因（Gadd45a，Gadd45g， Runx1和Selp）已经被证明在年轻小鼠中负调节HSC自我更新， 增强HSC自我更新，逆转偏斜的分化，从而使衰老的HSC恢复活力。拟议 研究将为HSC自我更新、分化和分化的潜在机制提供新的见解。 衰老在拟议的研究中发现的HSC再生的转化潜力将建立一个新的研究领域。 改善与年龄相关的疾病和促进健康的范例。