Identification of Genes Important for Rejuvenation of Aged Hematopoietic Stem Cells

摘要 造血干细胞具有广泛的自我更新和多向分化的能力。 终生维持所有血细胞的生产。然而，有充分证据表明，自我更新的能力 随着年龄的增长，HSCs的数量减少，并且老年HSCs在 淋巴生成的费用。这些与年龄相关的变化导致免疫力下降和 老年人患贫血和髓系恶性肿瘤的倾向增加。造血干细胞经历复杂的变化 在衰老过程中的基因表达。这些变化是否导致HSC老化等 重要的是，调制这些变化中的任何一种是否能够使旧的HSC恢复活力在很大程度上是未知的，并且 未被开发的。我们假设在衰老过程中HSCs中表达上调的某些基因 代表“老化”因素，并负责与年龄相关的HSC自我更新和 功能，而一类在HSC衰老过程中下调的基因是年轻的源泉类型 导致HSC衰老的因子及其表达减少。我们进一步假设耗尽了 关键的“老化”基因将使老的造血干细胞恢复活力。为了检验这些假设，我们将系统地确定 利用基于CRISPR的体内无偏基因筛查调控HSC自我更新和衰老的基因 有以下两个目标。在目标1中，我们将使用一个集中的慢病毒CRISPR文库来提供Cas9 核酸酶和21,268个单引导RNA(SgRNAs)靶向5,317个显示变化的小鼠基因 衰老过程中肝星状细胞的表达。从幼鼠体内分离的HSCs将被该CRISPR文库感染 携带不同sgRNAs的受感染的HSCs将被注射到干细胞耗尽的受鼠体内 在两轮移植中相互竞争地重新繁殖。更改的HSC自我更新和 功能将导致HSCs的浓缩或枯竭。通过分析特定sgRNAs的丰度 在HSCs和分化的B、T或髓系前后进行深度测序 移植后，我们将确定相应的基因，积极或消极地调节HSC自身 更新和差异化。在目标2中，我们将验证在目标1中确定的选定候选基因 它们在屏幕上的显著富集度或耗竭，并确定过程是否受 这些候选基因涉及HSC归巢、增殖、静止或分化。此外，我们 将测试在旧的HSCs中是否缺失候选的“衰老”基因和四个基因(Gadd45a，gadd45g， RUNX1和SELP)，已被证明对幼年小鼠的HSC自我更新具有负面调节作用 增强HSC的自我更新，逆转偏向分化，从而使陈旧的HSC恢复活力。建议数 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年轻化的翻译潜力将建立一个 改善年龄相关疾病和促进健康寿命的范例。

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.