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: 10088968
• 负责人: Karin Maria Hoffmeister
• 金额: $ 68.97万
• 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/10088968
• 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; GAG Gene; 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; base; cellular targeting; chemoradiation; chemotherapy; 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.

巨核细胞生成不足和血小板生成不足仍然是持续性血小板减少的主要原因 造血干细胞和祖细胞(HSPC)移植及放化疗后。血小板 需要输血以支持因此而增加输血反应、血小板风险的患者 同种免疫和输血相关病毒感染。重大工作的重点是确定 骨髓移植后促进血小板生成的最合适细胞和分子靶点 移植，化疗，并最大限度地发挥体外人血小板生产的功效 另一种增加供输血的血小板的方法。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，复苏和体外血小板产生受到阻碍 控制着血栓生成。巨核细胞(MK)驻留在骨髓中，维持持续产生 数十亿个循环中的血小板，以防止出血。最近的研究表明，MK还保持着 功能特定的BM利基，以支持偏向MK的HSPC。初步数据表明：1)MK偏向HSPC 表达异常高水平的α2，6-唾液酸(α2，6-SIA)，这是细胞固有的St6gal1mRNA所无法比拟的 表达；2)系统性ST6GAL1缺乏促进髓系倾斜的造血发育特征， 提示α2，6-唾液酸化是特定HSPC群体的检查点(项目2)；3)B4galt1缺失 在MKS中，β-1整合素过度激活，并意外地还调节硫酸肝素蛋白多糖(HS-PG) 和肝素酶在巨噬细胞中的表达，从而严重损害稳态和随后的血小板生成 清髓性损伤；4)肝素酶和糖胺多聚糖(GAG)模拟物可促进野生动物的血小板生成 类型和B4galt1-/-MKs，提示GAG在血小板生成中起作用。其中最重要的假设是 程序是“不同的细胞内源性和外源性糖链介导的机制调节维持， 造血细胞的分化和功能“。项目1将测试糖基化的具体假设 在三个目标中调节偏向于MK的造血干细胞和血小板生成：在目标1中，功能定义的偏向于MK的 造血干细胞将与项目2一起进行研究，特别是关于重度α2，6- 唾液酸化的细胞表面，尽管缺乏产生这种结构所必需的St6gal1表达。我们会 确定α2，6-SIA在MK偏向HSPC中的作用。根据已知的 半乳糖基转移酶β4GalT1在血栓形成中的作用，项目1还将研究β4GalT1， 糖胺多聚糖(GAG)/HS-PG在稳态和清髓剂后的血小板生成中的作用(目标2) 强调(目标3)使用新的共享“组学”与项目3和标准方法相结合。一个 我们还将探讨β4GalT1在调节HS PG MK表达中的作用。本项目 将发现增加血小板生成的新信息，并有助于了解其临床特征 由MK异常引起的。