Project 1: Megakaryocytes as Organizers of the Hematopoietic Environment

项目 1：巨核细胞作为造血环境的组织者

• 批准号: 10545012
• 负责人: Karin Maria Hoffmeister
• 金额: $ 68.91万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10545012
• 关键词: Abnormal megakaryocyte; Affect; Alloimmunization; Blood Platelets; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell surface; Cells; Clinical; Cluster Analysis; Data; Data Analyses; Defect; Development; Environment; Fluorouracil; Galactosyltransferases; Gene Expression Profile; Genomics; Glycobiology; Glycosaminoglycan Degradation Pathway; Glycosaminoglycans; Goals; Hematopoietic; Hematopoietic stem cells; Hemorrhage; Heparan Sulfate Proteoglycan; Heparin Lyase; Heterogeneity; Human; Hyperactivity; Impairment; In Vitro; Injury; Integrins; Investigation; Joints; Knowledge; Libraries; Maintenance; Mediating; Megakaryocytes; Megakaryocytopoieses; Messenger RNA; Methods; Molecular; Molecular Target; Mus; Myelogenous; Patients; Phenotype; Platelet Transfusion; Polysaccharides; Population; Production; Proteins; Proteome; Reaction; Recovery; Risk; Role; Sialic Acids; Source; Stem cell transplant; Stress; Structure; Structure-Activity Relationship; Sulfate; Surface; Syndrome; Testing; Therapeutic; Thrombocytopenia; Thrombopoiesis; Transfusion; Virus Diseases; Work; cellular targeting; chemoradiation; chemotherapy; comparison control; extracellular; genetic signature; glycosylation; heparanase; heparin proteoglycan; improved; in vivo; mRNA Expression; mimetics; mouse model; novel; prevent; programs; sialylation; single-cell RNA sequencing; transcriptome

Insufficient megakaryopoiesis and thrombopoiesis remain the main cause of persistent thrombocytopenia after hematopoietic stem and progenitor cell (HSPC) transplantation and after radiochemotherapy. Platelet transfusions are required to support patients with consequent increased risk of transfusion reactions, platelet alloimmunization, and transfusion-associated viral infections. Significant efforts are focused on identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow (BM) transplantation, chemotherapy, and to maximize the efficacy of in vitro human platelet production as an alternative method to increase the platelet supply for transfusion. Our ability to therapeutically optimize platelet recovery and in vitro platelet production is hindered by our paucity of knowledge of the molecular determinants that govern thrombopoiesis. Megakaryocytes (MKs) reside in the BM and maintain the continuous production of billions of circulating platelets in order to prevent bleeding. Recent work shows that MKs also maintain a functionally specific BM niche to support MK-biased HSPCs. Preliminary data suggest that: 1) MK-biased HSPCs express unusually high levels of α2,6-sialic acid (α2,6-Sia) which is unparalleled by cell intrinsic St6gal1 mRNA expression; 2) systemic ST6GAL1 deficiency promotes a myeloid skewed hematopoietic development profile, suggesting that α2,6-sialylation is a checkpoint of a particular HSPC population (Project 2); 3) B4galt1 deletion in MKs renders β1 integrin hyperactive and unexpectedly also regulates heparin sulfate proteoglycan (HS PG) and heparinase expression in MKs thereby severely impairing thrombopoiesis at steady state and following myeloablative injury; 4) Heparinase and glycosaminoglycan (GAG) mimetics improved thrombopoiesis in wild type and B4galt1-/- MKs, suggesting a role for GAGs in thrombopoiesis. The overarching hypothesis of this program is that “distinct cell-intrinsic and extrinsic glycan-mediated mechanisms regulate maintenance, differentiation, and function of hematopoietic cells”. Project 1 will test the specific hypothesis that glycosylation regulates MK-biased HSCs and thrombopoiesis in three aims: In Aim 1, a functionally defined MK-biased hematopoietic stem cell will be investigated together with Project 2, especially with respect to the heavily α2,6- sialylated cell surface despite the absence of St6gal1 expression necessary to generate this structure. We will establish the role of α2,6-Sia in MK-biased HSPCs. Based on the known requirement of the galactosyltransferase β4GalT1 in thrombopoiesis, Project 1 will also investigate the roles of β4GalT1, glycosaminoglycans (GAGs)/HS PG in thrombopoiesis at steady-state (Aim 2) and following myeloablative stress (Aim 3) using the novel combined shared “omics” together with Project 3 and standard approaches. A previously unknown role of β4GalT1 to regulate MK expression of HS PG will also be investigated. This Project will uncover new information to increase platelet production and help understand clinical conditions characterized by MK abnormalities.

巨核细胞生成和血小板生成不足仍然是持续性血小板减少症的主要原因， 造血干细胞和祖细胞（HSPC）移植和放化疗后。血小板 需要输血来支持患者，从而增加输血反应、血小板减少和血小板减少的风险。 同种异体免疫和输血相关的病毒感染。重要的努力集中在确定 最合适的细胞和分子靶点，以增强骨髓（BM）后的血小板生成 移植，化疗，并最大限度地提高体外人血小板生产的功效， 另一种方法，以增加血小板供应输血。我们在治疗上优化血小板的能力 由于我们缺乏对分子决定因素的了解， 控制血小板生成的基因巨核细胞（MK）驻留在BM中，并维持连续生产 数十亿的循环血小板来防止出血。最近的研究表明，MK也保持着 功能特异性BM小生境，以支持MK偏向的HSPC。初步数据表明：1）MK偏向的HSPCs 表达异常高水平的α 2，6-唾液酸（α 2，6-Sia），这是细胞内在St 6 gal 1 mRNA所无法比拟的 表达; 2）系统性ST 6 GAL 1缺乏促进骨髓偏斜的造血发育概况， 表明α 2，6-唾液酸化是特定HSPC群体的检查点（项目2）; 3）B4 galt 1缺失 在MK中使β1整联蛋白过度活跃，并且出乎意料地还调节硫酸肝素蛋白聚糖（HS PG） 和肝素酶的表达，从而严重损害稳态时的血小板生成， 清髓性损伤; 4）肝素酶和糖胺聚糖（GAG）模拟物改善野生型小鼠的血小板生成。 型和B4 galt 1-/-MK，表明GAG在血小板生成中的作用。这个最重要的假设是 程序是“不同的细胞内在和外在聚糖介导的机制调节维持， 造血细胞的分化和功能”。项目1将检验糖基化 在三个目标中调节MK偏向的HSC和血小板生成：在目标1中，功能上定义的MK偏向的 造血干细胞将与项目2一起研究，特别是关于α 2，6- 唾液酸化的细胞表面，尽管缺乏产生这种结构所必需的St 6 gal 1表达。我们将 确定α 2，6-Sia在MK偏性HSPC中的作用。根据已知的要求， 半乳糖基转移酶β 4GalT 1在血小板生成中的作用，项目1还将研究β 4GalT 1的作用， 在稳态（Aim 2）和清髓性清除后血小板生成中的糖胺聚糖（GAG）/HS PG 压力（目标3）使用新的组合共享“组学”与项目3和标准方法。一 还将研究β 4GalT 1在调节HS PG的MK表达中的先前未知的作用。这个项目 将揭示新的信息，以增加血小板的生产，并帮助了解临床条件的特点 MK异常。