Investigating the role of the Neogenin-1/Netrin-1 axis in aging and lineage bias of hematopoietic stem cells (HSCs)

项目摘要 造血干细胞（HSC）的定义是它们长期重建所有血液和免疫细胞的能力 在移植的宿主体内然而，HSC是异质性的，并且表现出不同程度的重建 对某些细胞谱系的潜在和偏见。HSC的这种多样性是两种内在变化的函数 例如获得性体细胞突变和表观遗传标记，以及外部影响，例如局部 血液中的骨髓形态因子和循环因子。从移植低 纯化的HSC的数量，存在至少两种类型的HSC，可通过它们对 不同的造血细胞系--偏向于骨髓的HSC，其对粒细胞和单核细胞的贡献更多 而不是T、B和NK细胞，以及平衡的HSC，其对所有谱系的贡献相等。在老化过程中， 造血干细胞的骨髓偏向性亚群克隆性扩增，并在旧骨髓中占主导地位，导致受损的造血干细胞。 血液和免疫产生以及白血病和白血病前期不良事件的风险增加。不过， 和骨髓偏向的HSC的身份，更不用说一种方法来前瞻性地将它们从平衡的HSC中分离出来。 HSC，未知。最近，我们观察到再生蛋白-1（Neo-1），一种细胞表面受体，标志着一部分 长期重建小鼠Hoxb 5 + HSC和人CD 34 + CD 38-CD 90 + CD 45 RA-HSC。串行 将Neo-1+和Neo-1-Hoxb 5 + HSC移植到亚致死剂量照射的宿主中显示，Neo-1标记 稳定的、骨髓偏向的小鼠Hoxb 5 + HSC，而Neo-1-Hoxb 5 + HSC是平衡的或淋巴偏向的。新- 1+ Hoxb 5 + HSC在体外也形成了更大的集落，并且比Neo-1-Hoxb 5 + HSC更常处于G2/S期。 此外，Netrin-1，一种化学引诱物分子和Neo-1的配体，在骨髓内皮细胞中表达。 细胞及其消耗导致外周血中髓样细胞数量减少， 野生型对照。鉴于这一证据，提出了两个具体目标：（1）确定功能和 小鼠和人衰老过程中Neo-1+和Neo-1-HSC之间的转录差异，以及（2） 确定骨髓定位并表征内皮Netrin-1的骨髓启动功能。 将测量小鼠中Neo-1+和Neo-1-HSC之间的扩增百分比和基因表达差异 人类随着年龄的增长。将老化的Neo-1+和Neo-1-Hoxb 5 + HSC移植到条件小鼠中， 将它们的总嵌合体和谱系嵌合体与我们来自年轻Neo-1+和Neo-1-Hoxb 5+移植物的数据进行比较。 此外，将对来自Hoxb 5-mCherry小鼠的骨髓进行成像以定位表达Netrin-1的细胞，并且 将评价Netrin-1缺失在内皮细胞中的作用。这些研究将提供新的信息 对造血干细胞异质性和干细胞重建的影响，并促进我们对造血中衰老的理解。

PROJECT ABSTRACT Hematopoietic stem cells (HSCs) are defined by their ability to long-term reconstitute all blood and immune cells in a transplanted host. However, HSCs are heterogeneous and exhibit different degrees of reconstitution potential and biases towards certain cell lineages. This diversity in HSCs is a function of both intrinsic changes in the cells, such as acquired somatic mutations and epigenetic marks, and external influences such as local bone marrow morphogens and circulating factors in the blood. It is evident from the transplantation of low numbers of purified HSCs that there exist at least two types of HSCs distinguishable by their contribution to different hematopoietic lineages—myeloid-biased HSCs, which contribute more to granulocytes and monocytes than to T, B, and NK cells, and balanced HSCs, which contribute equally to all lineages. During aging, the myeloid-biased subset of HSCs clonally expands and predominates the old bone marrow, leading to impaired blood and immune production and an increased risk of leukemia and preleukemic adverse events. Still, the origin and identity of the myeloid-biased HSCs, much less a method to prospectively separate them from balanced HSCs, is unknown. Recently, we observed that Neogenin-1 (Neo-1), a cell surface receptor, marks a fraction of long-term reconstituting mouse Hoxb5+ HSCs and human CD34+CD38-CD90+CD45RA- HSCs. Serial transplantation of Neo-1+ and Neo-1- Hoxb5+ HSCs into sublethally irradiated hosts reveals that Neo-1 marks stable, myeloid-biased mouse Hoxb5+ HSCs, while Neo-1- Hoxb5+ HSCs are balanced or lymphoid-biased. Neo- 1+ Hoxb5+ HSCs also formed larger colonies in vitro and were more often in G2/S than Neo-1- Hoxb5+ HSCs. Furthermore, Netrin-1, a chemoattractant molecule and ligand to Neo-1, is expressed in bone marrow endothelial cells and its depletion in mice results in reduced number of myeloid cells in the peripheral blood compared to wildtype controls. Given this evidence, two specific aims are proposed: (1) To identify the functional and transcriptional differences between Neo-1+ and Neo-1- HSCs during mouse and human aging, and (2) To determine the bone marrow localization and characterize the myeloid-priming function of endothelial Netrin-1. Percent expansion and gene expression differences will be measured between Neo-1+ and Neo-1- HSCs in mice and humans with age. Aged Neo-1+ and Neo-1- Hoxb5+ HSCs will be transplanted into conditioned mice and their total and lineage chimerism will be compared to our data from young Neo-1+ and Neo-1- Hoxb5+ transplants. Furthermore, bone marrow from Hoxb5-mCherry mice will be imaged to localize Netrin-1-expressing cells, and the effect of Netrin-1 deletion in endothelial cells will be evaluated. These studies will provide novel information on HSC heterogeneity and stem cell reconstitution and advance our understanding of aging in hematopoiesis.