Slit2-mediated expansion of primitive hematopoietic stem cell populations for tra

描述(由申请人提供)：资助机构：国家糖尿病、消化和肾脏疾病研究所(NIDDK)挑战领域-(11)再生医学特定挑战主题-11-DK-101促进消化系统、肝脏、胰腺、肾脏、血液和泌尿系统的再生和修复。3.项目名称：Slit2介导的原始造血干细胞群体的扩增用于移植和造血恢复造血干细胞(HSCs)将来自利基的信号与细胞内在调节程序整合在一起，以维持内源性HSC池。干细胞-生态位相互作用的调节影响整个淋巴造血系统，最终影响生物体，使这些途径的特征对于寻求涉及成人干细胞的新的治疗干预措施至关重要。基于近交系小鼠HSC绝对数的自然变异，我们启动了一项正向遗传学研究，揭示了5号染色体上的一个与C57B/L6(B6)和DBA/2(D2)小鼠的HSC数目相关的数量性状基因座。对5号染色体QTL同源菌株的表型分析证实，该区域的D2等位基因导致HSC数量增加2.4倍，而B6等位基因导致HSC数量减少1.7倍。我们利用基因芯片和RT-PCR技术，根据基因表达模式，筛选出CHR5基因QTL区域的HSC调控候选基因。我们发现，单个转录本Slit2在显著水平上存在差异表达，并且与HSC数量呈正相关。一种逆转录病毒被用来稳定地感染通常不表达Slit2的B6骨髓细胞，其中含有Slit2的GFP载体。感染的细胞被移植到B6宿主体内并在体内扩增12周，以确保在GFP+组分内重建完整的造血体系。体外定量研究表明，与空载体对照相比，Slit2的异位表达可使HSC数量增加4倍。这些结果证明，Slit2是一种新的小鼠造血调节因子，也是治疗多种人类疾病的潜在靶点。鉴于Sit/Robo信号在细胞间相互作用中的关键作用，以及已知的CXCL12/CXCR4轴的拮抗作用，我们预测Slit2介导的HSC调节是通过HSC-NICE相互作用的改变完成的。拟议的研究将确定Slit2的表达如何影响利基内的HSC以促进HSC池的扩大，以及Slit2的表达在HSC中是如何调节的。我们提出了三个目标：第一个目标是确定Slit2调节干细胞数量的机制。自我更新尤其令人感兴趣。第二个是确定Slit2基因的差异表达是如何调节的，特别是转录因子结合。第三个目标是研究调节Slit2表达的治疗潜力。将采用鼠标击倒模型。对干细胞在骨髓中定位的影响将使用成像技术来确定。 公共卫生相关性：这项建议调查了Slit2在调节骨髓中造血干细胞的数量和位置方面的作用。在三个特定的目标中，我们将研究SLIT2调节干细胞数量的机制(S)，特别是 注重自我更新。在第二个目标中，我们将通过以下方式来研究其机制(S Slit2在造血细胞中的表达受调控。在第三个目标中，治疗 将在基因敲除的小鼠模型中研究改变Slit2表达的可能性 使用成像技术定位骨髓中的干细胞。

DESCRIPTION (provided by applicant): Funding Agency: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Challenge Area - (11) Regenerative Medicine Specific challenge topic - 11-DK-101 Promote regeneration and repair in the digestive system, liver, pancrease, kidneys, hematologic, and urological system. 3. Project Title: Slit2-mediated expansion of primitive hematopoietic stem cell populations for transplantation and hematopoietic recovery Hematopoietic Stem Cells (HSCs) integrate signals from the niche with cell intrinsic regulatory programs to maintain the endogenous HSC pool. Regulation of stem cell-niche interactions influences the entire lymphohematopoietic system and ultimately the organism, making characterization of these pathways fundamental to the pursuit of novel therapeutic interventions involving adult stem cells. Based on natural variation in absolute numbers of HSCs in inbred mice, we initiated a forward genetic study that revealed a quantitative trait locus on chromosome 5 (Chr5) linked to the determination of HSC numbers in C57B/L6 (B6) and DBA/2 (D2) mice. Phenotypic analysis of strains congenic for the chromosome 5 QTL confirmed that D2 alleles in that region produce a 2.4-fold increase in HSC numbers while B6 alleles confer a 1.7-fold decrease in HSCs. We performed microarray and RT-PCR analysis to identify candidate HSC-regulatory genes in the Chr5 QTL region based on gene expression patterns. We found that a single transcript, Slit2, was differentially expressed at a significant level and positively correlated to HSC numbers. A retrovirus was used to stably infect B6 bone marrow cells, which normally do not express Slit2, with a Slit2-containing GFP vector. Infected cells were transplanted into B6 hosts and expanded in vivo for 12 weeks ensuring reconstitution of the complete hematopoietic hierarchy within the GFP+ fraction. In vitro quantification demonstrated that ectopic expression of Slit2 produces a 4-fold increase in HSC numbers relative to an empty vector control. These results provide evidence that Slit2 is a novel regulator of murine hematopoiesis and a potential target for therapeutic intervention in the treatment of a variety of human diseases. Given the pivotal role of Slit/Robo signaling in cell-cell interactions, and known antagonism of the CXCL12/CXCR4 axis we predict that Slit2- mediated regulation of HSCs is accomplished by alterations in HSC-niche interactions. Proposed studies will determine how Slit2 expression influences HSCs within the niche to promote expansion of the HSC pool and how Slit2 expression is regulated in HSCs. We propose three aims: The first is to determine the mechanisms by which SLIT2 regulates stem cell number. Self-renewal is of particular interest. The second is to determine how the differential expression of the Slit2 gene is regulated, with particular focus on transcription factor binding. The third aim will examine the therapeutic potential of modulating Slit2 expression. A mouse knockout model will be employed. The effects on stem cell localization within the bone marrow will be determined using imaging techniques. PUBLIC HEALTH RELEVANCE: This proposal investigates the role of Slit2 in regulating the number and location of hematopoietic stem cells within the bone marrow. In three specific aims we will investigate the mechanism(s) by which Slit2 regulates stem cell number, with special attention to self-renewal. In the second aim we will investigate the mechanism(s) by which Slit2 expression is regulated in hematopoietic cells. In the third aim the therapeutic potential of altering Slit2 expression will be investigated in a knockout mouse model and using imaging techniques to localize stem cells in the bone marrow.