Bone Marrow Spatial Transcriptomics to Enhance In Vitro Platelet Production

摘要 血小板输注在癌症治疗、骨髓移植、骨髓衰竭、 败血症、遗传性血小板紊乱等疾病。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变得同种异体致敏，使他们难以治愈。 去接受治疗。诱导多能干细胞(HiPSC)技术的发展引起了人们的兴趣 体外生产临床用血小板的可能性。这将提供相对无限的ON-1供应 需求血小板，包括个性化和人类白细胞抗原匹配/工程产品。然而，目前在体外 分化方案不能以足够高的效率产生实际使用的血小板。主要瓶颈 涉及其前体细胞--巨核细胞(Mk)产生血小板的最后阶段。这是目前 与体内相比，体外试验的效率至少降低了10到100倍。因此，克服这一障碍将 代表着在开发临床规模的血小板生产的体外方法方面向前迈出的重要一步。我们 假设当MKS接触骨髓血管时，关键的空间调节信号事件发生 它们通常在体内产生血小板的正弦壁龛。我们还假设目前的体外血小板 生产系统未能充分概括这些事件。进一步了解这些生理学 因此，发出事件信号是推进这一领域的关键。考虑到复杂的微环境，这些 事件发生时，现场检查这一问题是至关重要的。这项刺激血液学的目标 调查：新努力II(SIRE-II)建议采用新的空间转录技术进行研究 当MKS与血管血窦相互作用并开始产生血小板时发生的基因表达事件。它 将利用多重误差稳健的荧光原位杂交(MerFish)，这是一种提供 灵敏定量测量单个细胞中数万个基因的RNA表达 同时提供关于它们在组织切片中表达的空间信息。这将涉及到 利用小鼠系统和先导基因板开发用于骨髓血管血窦的水鱼。这 然后将扩展到整个转录组的规模，并应用于人类骨髓样本。功能界别 将利用人CD34+和HiPSCs在体外分化来探索选择有效途径的重要性 方法：研究方法。该项目的成功完成将对外地产生重大的积极影响，通过扩大 我们对触发巨噬细胞成熟并在其自然状态下产生血小板的生理信号事件的了解 微环境。由于血管血窦也是关键的造血干细胞(HSC)的生态位和水星鱼 捕获该领域内所有细胞的完整转录组基因表达测量，该项目还将 为HSC和血管生物学研究提供丰富的转录数据库。美人鱼的适应 福尔马林固定石蜡包埋的人骨髓活检标本也将使未来的原位基因 人类骨髓疾病的表达研究。

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.