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

Slit2介导的原始造血干细胞群扩增

• 批准号: 7824860
• 负责人: GARY VAN ZANT
• 金额: $ 43.88万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7824860
• 关键词: 5' Untranslated Regions; A Mouse; Alleles; American; Architecture; Area; Attention; Autoimmunity; Binding; Binding Sites; Biological Assay; Bone Marrow; Bone Marrow Cells; Breeding; Cell Communication; Cell Count; Cell Death; Cell physiology; Cells; Chromosomes, Human, Pair 5; Computer Simulation; Congenic Strain; Core Facility; CpG Islands; Defect; Drosophila sli protein; Ectopic Expression; Engraftment; Ensure; Equilibrium; Funding Agency; GTP Binding; Gene Expression; Generations; Genes; Genetic; Genetic Polymorphism; Genome; Goals; Healthcare; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Homing; Imagery; Imaging Techniques; Immigration; In Vitro; Inbred Mouse; Indium; Kentucky; Kidney; Knock-out; Knockout Mice; Link; Liver; Location; Luciferases; Mediating; Methylation; Migration Assay; Modeling; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Natural regeneration; Organism; Pancreas; Pancrease; Pathway interactions; Pattern; Physiological; Process; Quantitative Trait Loci; Recombinants; Recovery; Regenerative Medicine; Regulation; Regulator Genes; Relative (related person); Reporter; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Stem cells; System; Testing; Therapeutic; Therapeutic Intervention; Transcript; Transgenic Mice; Transgenic Organisms; Transplantation; Universities; Variant; adult stem cell; base; cancer therapy; clinically relevant; congenic; gastrointestinal system; human disease; improved; in vivo; interest; method development; mouse model; novel; novel therapeutic intervention; programs; promoter; public health relevance; reconstitution; repaired; self-renewal; sodium bisulfite; stem; stem cell niche; stem cell population; transcription factor; urologic; vector; vector control

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.

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 具体挑战主题 - 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扩增，用于移植和造血恢复造血干细胞 (HSC) 将微生境信号与细胞内在调节程序相结合，以维持内源性 HSC 库。干细胞生态位相互作用的调节影响整个淋巴造血系统并最终影响生物体，使得这些途径的表征对于寻求涉及成体干细胞的新型治疗干预措施至关重要。基于近交系小鼠 HSC 绝对数量的自然变异，我们发起了一项正向遗传学研究，揭示了 5 号染色体 (Chr5) 上的数量性状位点与 C57B/L6 (B6) 和 DBA/2 (D2) 小鼠 HSC 数量的测定相关。对 5 号染色体 QTL 同源菌株的表型分析证实，该区域的 D2 等位基因使 HSC 数量增加 2.4 倍，而 B6 等位基因使 HSC 数量减少 1.7 倍。我们进行了微阵列和 RT-PCR 分析，根据基因表达模式识别 Chr5 QTL 区域中的候选 HSC 调节基因。我们发现单个转录本 Slit2 具有显着水平的差异表达，并且与 HSC 数量呈正相关。使用逆转录病毒和含有 Slit2 的 GFP 载体稳定感染通常不表达 Slit2 的 B6 骨髓细胞。受感染的细胞被移植到 B6 宿主中并在体内扩增 12 周，确保在 GFP+ 部分内重建完整的造血层次结构。体外定量表明，相对于空载体对照，Slit2 的异位表达使 HSC 数量增加 4 倍。这些结果证明 Slit2 是一种新型的小鼠造血调节因子，也是多种人类疾病治疗干预的潜在靶标。鉴于 Slit/Robo 信号传导在细胞间相互作用中的关键作用以及已知的 CXCL12/CXCR4 轴拮抗作用，我们预测 Slit2 介导的 HSC 调节是通过改变 HSC-生态位相互作用来实现的。拟议的研究将确定 Slit2 表达如何影响生态位内的 HSC，以促进 HSC 池的扩张，以及 Slit2 表达如何在 HSC 中受到调节。我们提出三个目标：第一个是确定 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，特别关注转录因子结合。第三个目标是检查调节 Slit2 表达的治疗潜力。将采用小鼠敲除模型。将使用成像技术确定对骨髓内干细胞定位的影响。 公共健康相关性：该提案研究了 Slit2 在调节骨髓内造血干细胞的数量和位置方面的作用。在三个具体目标中，我们将研究 Slit2 调节干细胞数量的机制，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研究机制： Slit2 的表达在造血细胞中受到调节。第三个目标是治疗 将在敲除小鼠模型中研究改变 Slit2 表达的潜力 使用成像技术定位骨髓中的干细胞。