Hemolysis and the Hematopoietic Niche

摘要 镰状细胞病（SCD）是一种主要的医疗负担，治疗选择有限。当前可用 治疗仅限于输血、羟基脲和L-谷氨酰胺。随着人们对造血系统的兴趣日益浓厚， 干细胞（HSC）移植和基因治疗作为SCD的治疗选择，了解溶血性 应激改变骨髓（BM）微环境对于开发适当的治疗药物至关重要。 战略布局在目标1中，我们将研究溶血对骨髓龛（包括间充质干细胞）的影响。 细胞（MSC），负责维持HSC，和成红细胞岛（EBI），红细胞生成的生态位。 在目标2中，我们将定义溶血对红系祖细胞/前体细胞的影响机制。我们 进一步建议确定是否用输血、血红素清除剂血红素结合素或两者联合治疗 能改善骨髓间充质干细胞和EBI巨噬细胞的功能。我们的总体假设是游离血红素导致 BM造血生态位和造血干/祖细胞的功能障碍，这可以在 部分通过血液结合素和/或输血治疗。首先，我们将研究溶血对骨髓间充质干细胞的影响， 调节造血的能力。我们将评估许多MSC干细胞的特性，并研究如何 这些变化反过来又会影响HSC的活化状态、ROS的积累和DNA损伤。接下来，我们将定义 由于溶血导致红细胞生成活性受损的机制， EBI形成、CFU-E集落形成和去核。这些研究通过SCD小鼠模型实现， 塔尔小鼠模型，Epor-eGFP敲入小鼠模型，靶向骨髓间充质干细胞的小鼠模型，我们最近的 Epor+ EBI巨噬细胞的鉴定，抗小鼠Epor和抗人EPOR抗体的验证 以及我们已经开发出的在不同发育阶段纯化红系细胞并定量 鼠和人BM终末红细胞分化。这些研究将提供全面的 机制的理解，溶血对骨髓造血生态位的影响，并有助于 开发靶向BM小生境细胞的新型治疗剂，并最终改善 SCD患者

ABSTRACT Sickle cell disease (SCD) is a major healthcare burden with limited treatment options. Currently available treatments are limited to transfusions, hydroxyurea and L-glutamine. With increasing interest in hematopoietic stem cell (HSC) transplantation and gene therapy as treatment options for SCD, understanding how hemolytic stress alters the bone marrow (BM) microenvironment is critical for the development of appropriate therapeutic strategies. In Aim 1, we will investigate the effects of hemolysis on the BM niches including mesenchymal stem cells (MSCs), responsible for maintaining HSCs, and erythroblastic islands (EBIs), the niche for erythropoiesis. In Aim 2, we will define the mechanisms for the effects of hemolysis on erythroid progenitors/precursors. We further propose to determine whether therapy with transfusion, heme scavenger hemopexin or the combination can improve the function of MSCs and EBI macrophages. Our overall hypothesis is that free heme leads to dysfunction of the BM hematopoietic niche and hematopoietic stem/progenitor cells, which can be alleviated in part by hemopexin and/or transfusion therapy. First, we will investigate the effects of hemolysis on the BM MSC ability to regulate hematopoiesis. We will evaluate numerous MSC stem cell properties and investigate how alterations may in turn affect HSC activation state, accumulation of ROS and DNA damage. Next, we will define the mechanisms for the impaired erythropoietic activity due to hemolysis, including mechanisms for defective EBIs formation, CFU-E colony formation and enucleation. These studies are enabled by the SCD mouse model, Thal mouse model, Epor-eGFP knockin mouse model, mouse models targeting BM MSCs, our recent identification of Epor+ EBI macrophages, validation of both anti-mouse Epor and anti-human EPOR antibodies as well as the methods we have developed to purify erythroid cells at distinct developmental stage and to quantify both murine and human BM terminal erythroid differentiation. These studies will provide a comprehensive mechanistic understanding of the effects of hemolysis on BM hematopoietic niches, and contribute to the development of novel therapeutics targeting the BM niche cells and ultimately improve treatment options for patients with SCD.