Bone Marrow Spatial Transcriptomics to Enhance In Vitro Platelet Production

骨髓空间转录组学可增强体外血小板生产

• 批准号: 10457431
• 负责人: ALAN B. CANTOR
• 金额: $ 35.24万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10457431
• 关键词: Biogenesis; Biology; Biopsy Specimen; Blood Cells; Blood Circulation; Blood Platelet Disorders; Blood Platelets; Blood Vessels; Bone Marrow; Bone Marrow Diseases; Bone Marrow Transplantation; Bone marrow biopsy; Bone marrow failure; CD34 gene; Cantor; Cells; Chronic; Clinical; Complex; Databases; Development; Discontinuous Capillary; Disease; Dysmyelopoietic Syndromes; Endothelial Cells; Engineering; Event; Femur; Fluorescent in Situ Hybridization; Formalin; Future; Gene Expression; Generations; Genes; Genetic; Genetic Diseases; Genetic Transcription; Goals; Hematological Disease; Hematology; Hematopoietic stem cells; Human; In Situ; In Vitro; Inherited; Investigation; Knowledge; Life; Measurement; Measures; Megakaryocytes; Megakaryocytopoieses; Methods; Mus; Outcome Study; Paraffin Embedding; Pathway interactions; Patients; Pharmacology; Physiological; Platelet Transfusion; Play; Process; Production; Protocols documentation; Refractory; Regenerative Medicine; Research; Research Personnel; Resources; Role; Sampling; Savings; Sepsis; Side; Signal Pathway; Signal Transduction; Slice; Source; Stromal Cells; System; Techniques; Technology; Testing; Time; Tissues; Transfusion; Tube; Vascular Endothelial Cell; Vascular Endothelium; cancer therapy; differentiation protocol; gene expression database; hematopoietic stem cell niche; in vivo; induced pluripotent stem cell; leukemia; new technology; novel; precursor cell; premature; programs; response; single cell analysis; stem cells; tool; transcriptome; transcriptomics

ABSTRACT Platelet transfusions play life-saving roles for cancer therapy, bone marrow transplantation, bone marrow failure, sepsis, genetic platelet disorders, and other diseases. However, the limited shelf life of donor platelets causes frequent shortages. In addition, multiply transfused patients often become allosensitized making them refractory to treatment. The development of induced pluripotent stem cell (hIPSC) technology has raised the intriguing possibility of producing platelets in vitro for clinical use. This would provide a relatively limitless supply of on- demand platelets, including personalized and HLA matched/engineered products. However, current in vitro differentiation protocols fail to generate platelets at high enough efficiency for practical use. The major bottleneck involves the final stages of platelet production from their precursor cell, the megakaryocyte (Mk). This is currently at least 10 to 100-fold less efficient in vitro compared to in vivo. Overcoming this obstacle would therefore represent a major step forward in developing in vitro methods for clinical scale platelet production. We hypothesize that key spatially regulated signaling events occur when Mks engage the bone marrow vascular sinusoidal niche, where they normally produce platelets in vivo. We also hypothesize that current in vitro platelet production systems fail to adequately recapitulate these events. Further understanding these physiologic signaling events is therefore key to advancing this field. Given the complex microenvironment in which these events occur, it is critical to examine this problem in situ. The objective of this Stimulating Hematology Investigation: New Endeavors II (SHINE-II) proposal is to adapt new spatial transcriptomic technology to study gene expression events that occur when Mks interact with vascular sinusoids and begin producing platelets. It will utilize Multiplexed Error-robust Fluorescence In Situ Hybridization (MERFISH), a technique that provides sensitive and quantitative measurements of RNA expression of tens of thousands of genes in single cells while at the same time providing spatial information regarding their expression in tissue slices. This will involve developing MERFISH for the bone marrow vascular sinusoid using murine systems and pilot gene panels. This will then be extended to whole-transcriptome scale and applied to human bone marrow samples. The functional importance of select validated pathways will be explored using human CD34+ and hIPSCs in vitro differentiation methods. Successful completion of the project will have a significant positive impact on the field by expanding our knowledge of the physiologic signaling events that trigger Mks to mature and produce platelets in their natural microenvironment. As the vascular sinusoid is also a key hematopoietic stem cell (HSC) niche and MERFISH captures whole transcriptome gene expression measurements on all cells within the field, this project will also provide a rich transcriptomic database for studies of HSC and vascular biology. The adaptation of MERFISH to human formalin fixed paraffin embedded bone marrow biopsy specimens will also enable future in situ gene expression studies of human bone marrow disorders.

抽象的 血小板输注在癌症治疗、骨髓移植、骨髓衰竭、 败血症、遗传性血小板疾病和其他疾病。然而，捐献者血小板的保质期有限，导致 经常出现短缺。此外，多次输血的患者通常会出现异体过敏，从而导致难治性 来治疗。诱导多能干细胞（hIPSC）技术的发展引起了人们的关注。 体外生产用于临床的血小板的可能性。这将提供相对无限的供应 需求血小板，包括个性化和 HLA 匹配/工程产品。然而，目前体外 分化方案无法以足够高的效率产生可供实际使用的血小板。主要瓶颈 涉及从其前体细胞巨核细胞（Mk）产生血小板的最后阶段。这是目前 与体内相比，体外效率至少低 10 至 100 倍。因此，克服这一障碍将 代表着开发用于临床规模血小板生产的体外方法向前迈出的重要一步。我们 假设当 Mks 参与骨髓血管时，会发生关键的空间调节信号事件 正弦生态位，它们通常在体内产生血小板。我们还假设目前的体外血小板 生产系统无法充分重现这些事件。进一步了解这些生理 因此，信号事件是推动这一领域发展的关键。考虑到这些物质所处的复杂微环境 当事件发生时，现场检查这个问题至关重要。刺激血液学的目的 调查：New Endeavors II (SHINE-II)提案是采用新的空间转录组技术来研究 当 Mks 与血管正弦相互作用并开始产生血小板时发生的基因表达事件。它 将利用多重抗差错荧光原位杂交 (MERFISH)，该技术可提供 对单细胞中数万个基因的 RNA 表达进行灵敏和定量测量，同时 同时提供有关它们在组织切片中表达的空间信息。这将涉及到 使用小鼠系统和先导基因组开发用于骨髓血管正弦波的 MERFISH。这 然后将扩展到全转录组规模并应用于人类骨髓样本。功能性的 将使用人 CD34+ 和 hIPSC 体外分化来探索选择经过验证的途径的重要性 方法。该项目的成功完成将扩大该领域的影响力，从而对该领域产生重大的积极影响。 我们对触发 Mks 成熟并自然产生血小板的生理信号事件的了解 微环境。由于血管正弦也是一个关键的造血干细胞 (HSC) 生态位，MERFISH 捕获该领域内所有细胞的全转录组基因表达测量，该项目还将 为HSC和血管生物学的研究提供丰富的转录组数据库。 MERFISH 的适应 人类福尔马林固定石蜡包埋的骨髓活检标本也将使未来的原位基因成为可能 人类骨髓疾病的表达研究。