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。目前可用 治疗仅限于输血、羟基尿素和L-谷氨酰胺。人们对造血学的兴趣与日俱增 干细胞(HSC)移植和基因治疗作为SCD的治疗选择，了解如何溶血 应激改变骨髓(BM)微环境对开发合适的治疗方法至关重要 战略。在目标1中，我们将研究溶血对包括间充质干细胞在内的骨髓壁龛的影响。 负责维持HSCs的细胞(MSCs)和红系造血岛(EBI)，这是红血球生成的利基。 在目标2中，我们将确定溶血对红系祖细胞/前体细胞影响的机制。我们 进一步建议确定是否使用输血、血红素清除剂血凝素或联合治疗 能改善MSCs和EBI巨噬细胞的功能。我们的总体假设是游离的亚铁血红素导致 骨髓造血龛和造血干/祖细胞功能障碍，可以缓解 部分通过血液凝集素和/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 改变可能反过来影响HSC的激活状态、ROS的积累和DNA损伤。接下来，我们将定义 溶血引起的红细胞生成功能受损的机制，包括缺陷的机制 EBIS形成、CFU-E集落形成和去核。这些研究是通过SCD小鼠模型实现的， Thal小鼠模型、EPOR-EGFP敲门小鼠模型、BM MSCs靶向小鼠模型、我们最新的 EPOR+EBI巨噬细胞的鉴定及抗鼠EPOR和抗人EPOR抗体的验证 以及我们开发的纯化不同发育阶段的红系细胞并对其进行量化的方法 小鼠和人骨髓均为红系终末分化细胞。这些研究将提供一个全面的 从机制上理解溶血对骨髓造血生态位的影响，并有助于 开发针对BM利基细胞的新疗法，并最终改进治疗方案 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.