Systematic identification of hematopoietic stem cell expansion factors

造血干细胞扩增因子的系统鉴定

基本信息

批准号：
9754241
负责人：
Jiwang Zhang
金额：
$ 37.09万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9754241
关键词：
ANGPTL2 gene Address Adult Agonist Biological Assay Bone Marrow Cell Count Cell Culture Techniques Cell Proliferation Cells Clinical Cytokine Gene Disease Endothelial Cells Expression Profiling Flow Cytometry Fluorouracil Frequencies Gene Expression Genes Genomics Hematopoiesis Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Human IGFBP2 gene In Vitro Life Long-Term Effects Mediating Mesenchymal Stem Cells Modeling Molecular Mus Mutation Neonatal Pathologic Phenotype Problem Solving RNA Sequences Resources Signal Transduction Solid Neoplasm Stimulus System Techniques Time Umbilical Cord Blood WNT Signaling Pathway beta catenin cell behavior cytokine effective therapy hematopoietic stem cell expansion hematopoietic stem cell quiescence hematopoietic stem cell self-renewal improved leukemia peripheral blood planar cell polarity postnatal development receptor

项目摘要

Hematopoietic stem cell (HSC) transplantation is the most effective therapy for life-threatening hematopoietic diseases such as leukemia and many solid tumors. However such therapy is significantly limited by the shortage of HSC resources. In vitro HSC expansion is believed to be the most effective and applicable strategy to address this problem. Despite significant efforts toward this end, our ability to expand HSCs remains clinically unsuccessful because the key factors that promote HSC self-renewal have not been identified. Two reasons might explain why we failed to do so: 1) the levels of the factor(s) in bone marrow (BM) are very low which cannot be detected by previous used techniques; 2) a proper combination of factors might be required which needs a reliable assay to determine. HSC behaviors are tightly controlled by specialized BM niche cells and their secreted factors. We found that in mice, HSCs in the BM expand by approximately 100 times during the 1-2 weeks of postnatal development. We also detected an approximately 4-fold increase in functional HSC numbers on day 4 following a single treatment with 5FU. By taking the advantage of these two HSC expansion models, we examined the dynamic changes of niche cells by flow cytometry and their gene expression profiles by RNA-sequence. Such assays allow us to sensitively detect the changes in lower frequency of niche cells at 0.01% level and lower expressing genes. We specifically searched for the niche cells and their secreted factors that were increased prior to HSC expansion in both models. We speculate that such niche cells and factors might be potential candidates of HSC stimuli. In addition, we also developed a very simple and reliable ex vivo assay to evaluate the expansion of functional HSCs. We found that the dynamic changes of the CD31high endothelial cells and CD140a+CD51+ mesenchymal stem cells (MSCs), as well as a group of 78 cytokine-encoding genes expressed by these two types of niche cells are closely correlated with HSC expansion in both models. Among these cytokines, by functional analysis, we have already shown that R-spondin 2 (Rspo2) and Rspo3, Wnt agonists, can promote 15-20-fold expansion of murine HSCs in a Wnt-dependent manner. We intend to study the molecular mechanism by which Rspo2 stimulates HSC expansion (Aim 1). We will further characterize the phenotype of CD140a+CD51+ MSCs and CD31hi cells and determine the molecular mechanism by which these two types of niche cells synergistically promote HSC expansion (Aim 2). We want to determine how long the Rspo2 can sustain HSC expansion in vitro and whether Rspo2 induces genomic mutations in HSCs. We also want to evaluate whether Rspo2, CD140a+CD51+ MSCs and CD31hi cells can promote human cord blood HSCs and mobilized peripheral blood HSCs (Aim 3). We expect to identify the key stimuli of HSC self-renewal and develop an improved in vitro HSC expansion system which can be used for clinical HSC transplantation therapy. The results expected from this study will also help us to understand the molecular mechanism by which HSC activity is regulated in both normal and pathological hematopoiesis.

造血干细胞（HSC）移植是危及生命的最有效治疗方法造血系统疾病，例如白血病和许多实体瘤。然而，这种疗法有很大的局限性由于HSC资源短缺。体外HSC扩增被认为是最有效和最适用的解决这个问题的策略。尽管为此付出了巨大努力，但我们扩展 HSC 的能力仍然存在临床上不成功是因为促进HSC自我更新的关键因素尚未确定。二原因可能可以解释为什么我们未能这样做：1）骨髓（BM）中的因子水平非常低以前使用的技术无法检测到的； 2）可能需要适当的因素组合这需要可靠的测定来确定。 HSC 的行为受到专门的 BM 生态位细胞的严格控制及其分泌因子。我们发现，在小鼠中，BM 中的 HSC 在复制过程中扩增了大约 100 倍。产后1-2周发育。我们还检测到功能性 HSC 增加了约 4 倍单次 5FU 治疗后第 4 天的数字。通过利用这两个 HSC 扩展模型中，我们通过流式细胞仪检查了微环境细胞的动态变化及其基因表达谱通过RNA序列。此类测定使我们能够灵敏地检测低频率利基细胞的变化 0.01%水平和较低表达基因。我们专门寻找利基细胞及其分泌因子在 HSC 扩展之前，这两种型号均有所增加。我们推测这些利基细胞和因子可能是 HSC 刺激的潜在候选者。此外，我们还开发了一种非常简单且可靠的离体测定法评估功能性 HSC 的扩展。我们发现CD31高内皮细胞的动态变化和 CD140a+CD51+ 间充质干细胞 (MSC)，以及一组 78 个细胞因子编码基因在两种模型中，这两种类型的微环境细胞所表达的与 HSC 的扩增密切相关。之中这些细胞因子，通过功能分析，我们已经表明 R-spondin 2 (Rspo2) 和 Rspo3，Wnt 激动剂，可以以Wnt依赖性方式促进小鼠HSC扩增15-20倍。我们打算研究分子 Rspo2 刺激 HSC 扩张的机制（目标 1）。我们将进一步表征 CD140a+CD51+ MSC 和 CD31hi 细胞，并确定这两种类型的分子机制生态位细胞协同促进 HSC 扩增（目标 2）。我们想要确定 Rspo2 可以持续多久体外维持 HSC 扩增以及 Rspo2 是否诱导 HSC 基因组突变。我们也想评价一下 Rspo2、CD140a+CD51+ MSCs和CD31hi细胞能否促进人脐带血HSCs动员外周血 HSC（目标 3）。我们期望确定 HSC 自我更新的关键刺激因素并开发一种改进的体外HSC扩增系统可用于临床HSC移植治疗。结果这项研究预计也将有助于我们了解 HSC 活性的分子机制在正常和病理性造血过程中均受到调节。