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术后持续性血小板减少的主要原因 造血干细胞和祖细胞（HSPC）移植以及放化疗后。血小板 需要输血来支持患者，从而增加输血反应、血小板的风险 同种免疫和输血相关病毒感染。重大努力的重点是确定 继骨髓（BM）之后增强血小板生成的最合适的细胞和分子靶标 移植、化疗以及最大限度地提高体外人血小板生产的功效 增加输血血小板供应的替代方法。我们治疗优化血小板的能力 由于我们对分子决定因素知识的缺乏，阻碍了恢复和体外血小板生产 控制血小板生成。巨核细胞 (MK) 驻留在 BM 中并维持持续生产 数十亿的循环血小板以防止出血。最近的工作表明 MK 还保持 功能特定的 BM 利基来支持偏向 MK 的 HSPC。初步数据表明：1）偏向 MK 的 HSPC 表达异常高水平的 α2,6-唾液酸 (α2,6-Sia)，这是细胞内在 St6gal1 mRNA 无法比拟的 表达; 2) 系统性 ST6GAL1 缺乏会促进骨髓造血发育特征的倾斜， 表明 α2,6-唾液酸化是特定 HSPC 群体的检查点（项目 2）； 3) B4galt1缺失 MK 中的 β1 整合素过度活跃，并且意外地还调节硫酸肝素蛋白聚糖 (HS PG) MK 中的肝素酶表达，从而严重损害稳态和后续状态下的血小板生成 清髓性损伤； 4) 肝素酶和糖胺聚糖 (GAG) 模拟物可改善野生状态下的血小板生成 型和 B4galt1-/- MK，表明 GAG 在血小板生成中发挥作用。本研究的总体假设 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将检验糖基化的具体假设 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 造血干细胞将与项目2一起研究，特别是关于α2,6- 尽管缺乏生成这种结构所必需的 St6gal1 表达，但唾液酸化的细胞表面仍然存在。我们将 确定 α2,6-Sia 在 MK 偏向 HSPC 中的作用。根据已知的要求 半乳糖基转移酶β4GalT1在血小板生成中的作用，项目1还将研究β4GalT1的作用， 糖胺聚糖 (GAG)/HS PG 在稳态（目标 2）和清髓性血小板生成中的作用 使用新颖的将共享“组学”与项目 3 和标准方法相结合的压力（目标 3）。一个 β4GalT1 调节 HS PG MK 表达的先前未知的作用也将被研究。本项目 将发现增加血小板生成的新信息并帮助了解特征性的临床病症 MK 异常。