Systematic identification of hematopoietic stem cell expansion factors

造血干细胞移植是治疗危及生命的最有效的方法。 血液系统疾病，如白血病和许多实体肿瘤。然而，这种疗法是非常有限的。 由于HSC资源的短缺。体外扩增HSC被认为是最有效和最适用的方法 解决这一问题的战略。尽管为此目的作出了重大努力，但我们扩大HSC的能力仍然 临床上不成功，因为促进HSC自我更新的关键因素尚未确定。二 我们没有做到这一点的原因可能是：1)骨髓中S因子的水平很低 这是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 需要一种可靠的检测方法来确定。HSC的行为受到专门的BM利基细胞的严格控制 以及他们的秘密因素。我们发现，在小鼠体内，骨髓中的HSCs在 出生后1-2周发育。我们还检测到功能性HSC大约增加了4倍 5FU单次治疗后第4天的数字。通过利用这两个HSC扩展 通过流式细胞仪检测细胞的动态变化及其基因表达谱 经RNA序列测定。这样的分析使我们能够灵敏地检测到较低频率的壁龛细胞在 0.01%的水平和较低的表达基因。我们专门寻找了壁龛细胞和它们的分泌因子 在两个型号的HSC扩展之前都有所增加。我们推测，这些利基细胞和因子可能 成为HSC刺激的潜在候选者。此外，我们还开发了一种非常简单可靠的体外试验来 评估功能性肝星状细胞的扩展。我们发现CD31高表达的内皮细胞的动态变化 和CD140a+CD51+间充质干细胞(MSCs)，以及一组78个细胞因子编码基因 在这两种模型中，这两种细胞的表达与HSC的扩增密切相关。其中 这些细胞因子，通过功能分析，我们已经表明，R-响应蛋白2(Rsp2)和Rsp3，Wnt激动剂， 能以Wnt依赖的方式促进小鼠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)。我们将进一步描述其表型 CD140a+CD51+MSCs和CD31hi细胞，并确定这两种类型的 利基细胞协同促进肝星状细胞扩增(目标2)。我们想要确定Rsp2能持续多久 维持HSC的体外扩增，以及Rsp2是否诱导HSC的基因组突变。我们还想评估 Rspo2、CD140a+CD51+MSCs和CD31hi细胞能否促进人脐血HSCs并动员 外周血HSCs(目标3)。我们希望确定HSC自我更新的关键刺激因素，并制定 改良的HSC体外扩增系统可用于临床的HSC移植治疗。结果是 这项研究也将有助于我们理解HSC活性的分子机制 在正常和病理性的造血中都有调节作用。

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.