Megakaryocyte Transcription Factor Activation to Enhance In Vitro Platelet Production from Human IPSCs

巨核细胞转录因子激活可增强人 IPSC 的体外血小板生成

• 批准号: 9276771
• 负责人: ALAN B. CANTOR
• 金额: $ 51.04万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-10 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276771
• 关键词: Anabolism; Autoimmune Diseases; Autoimmune Process; Biogenesis; Blood Component Removal; Blood Platelet Disorders; Blood Platelets; Blood Vessels; Bone Marrow; Bone Marrow Transplantation; Calcineurin; Clinical; Codon Nucleotides; Complex; Development; Embryo; Embryonic Development; Engineering; Event; FLI1 gene; Friends; GATA1 gene; Genetic; Health; Human; In Situ; In Vitro; Infection; Inherited; Knowledge; Life; Literature; Mediating; Megakaryocytes; Megakaryocytopoieses; Membrane; Methods; Multiprotein Complexes; Mus; Outcome; Outcome Study; PTPN11 gene; Pancytopenia; Pathway interactions; Patients; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Platelet Transfusion; Play; Process; Production; Protein Dephosphorylation; Protocols documentation; RUNX1 gene; Refractory; Role; Savings; Sepsis; Serine; Signal Pathway; Signal Transduction; Site; Techniques; Technology; Testing; Thrombopoiesis; Tyrosine Phosphorylation; Work; base; cancer therapy; gene repression; genetic manipulation; in vivo; induced pluripotent stem cell; kinase inhibitor; precursor cell; progenitor; public health relevance; src-Family Kinases; stem; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): Platelet transfusions are a life-saving treatment for health conditions related to cancer therapy, bone marrow transplantation, bone marrow failure, sepsis, genetic platelet disorders, and some autoimmune conditions. Currently, over 2 million apheresis platelet units and over 4 million pooled platelet concentrate units are transfused in the U.S. each year. However the limited shelf life of donor platelets leads to frequent shortages. Moreover, multiply transfused patients often become refractory due to allosensitization. The advent of human embryonic stem (hES) and induced pluripotent stem cell (hIPSC) technology has raised the possibility of producing platelets in vitro for clinical use. This would allow for a essentially limitless supply of on-demand platelets and could be engineered to overcome allosensitization problems. However, current protocols are hampered by low platelet production efficiency. The major bottleneck is at the level of terminal megakaryocyte (Mk) maturation and platelet release, which is about 1000-fold lower in vitro compared to primary Mks in vivo. The major objective of this proposal is to further understand the physiologic mechanisms that regulate terminal thrombopoiesis and to apply these to enhance hIPSC in vitro platelet production. Terminal Mk maturation requires the action of a small group of key transcription factors (RUNX1, GATA1, Friend of GATA1, and FLI1) that physically associate with one another during differentiation. This drives high- level activation or repression of genes involved in polyploidization, platelet membrane genesis, platelet constituent biosynthesis and proplatelet formation. We hypothesize that key signaling events control the assembly of this "Mk enhancesome complex", and can be exploited to enhance IPSC based platelet production. We have previously identified key signaling events that regulate differentiation-dependent interaction among these factors. This includes src-family kinase (SFK) mediated tyrosine phosphorylation of RUNX1 and serine phosphorylation of FLI1 at codon 10. These findings are supported by work from other groups showing marked enhancement of primary murine and human Mk maturation by SFK inhibitors. The aims of this proposal are to further elaborate these signaling pathways and to determine the extent to which their pharmacologic and/or genetic manipulation enhances hIPSC-derived Mk maturation and platelet release, while preserving platelet function. In addition, we propose to apply the newly developed technique Fluorescent in situ RNA seq (FISSEQ) for non-biased discovery of additional signaling pathways that are engaged when Mk progenitors physically interact with the vascular sinusoidal niche, the physiologic site of terminal Mk maturation and platelet release. The expected outcome of this project is the elucidation of specific physiologic signaling pathways that can be manipulated to increase hIPSC platelet production efficiency.

 描述（由申请人提供）：血小板输注是与癌症治疗、骨髓移植、骨髓衰竭、败血症、遗传性血小板疾病和某些自身免疫性疾病相关的健康状况的挽救生命的治疗。目前，超过200万个单采血小板单位和超过400万个汇集的血小板浓缩物单位被输注在 美国每年.然而，有限的保存期限的供体血小板导致经常短缺。此外，多次输血的患者往往由于同种异体致敏而变得难治。人类胚胎干细胞（hES）和诱导多能干细胞（hIPSC）技术的出现提高了体外生产血小板用于临床的可能性。这将允许基本上无限的按需血小板供应，并且可以被工程化以克服同种异体致敏问题。然而，目前的方案受到低血小板生产效率的阻碍。主要的瓶颈是在终末巨核细胞（Mk）成熟和血小板释放的水平，这是约1000倍低于在体外相比，在体内的主要Mk。该提案的主要目的是进一步了解调节终末血小板生成的生理机制，并将其应用于增强hIPSC体外血小板生成。终末Mk成熟需要一小组关键转录因子（RUNX 1，GATA 1，Friend of GATA 1和FLI 1）的作用，这些转录因子在分化过程中彼此物理关联。这驱动参与多倍化、血小板膜发生、血小板成分生物合成和前血小板形成的基因的高水平激活或抑制。我们假设关键信号事件控制这种“Mk增强体复合物”的组装，并且可以被利用来增强基于IPSC的血小板产生。我们先前已经确定了调节这些因子之间的分化依赖性相互作用的关键信号传导事件。这包括src家族激酶（SFK）介导的RUNX 1的酪氨酸磷酸化和FLI 1在密码子10处的丝氨酸磷酸化。这些发现得到了来自其他组的工作的支持，这些工作显示SFK抑制剂显著增强了原代鼠和人Mk成熟。本提案的目的是进一步阐述这些信号通路，并确定其药理学和/或遗传操作在多大程度上增强hIPSC衍生的Mk成熟和血小板释放，同时保留血小板功能。此外，我们建议应用新开发的技术荧光原位RNA测序（FISSEQ）的非偏见的发现额外的信号转导途径，从事时，Mk祖细胞物理相互作用的血管窦状隙，终端Mk成熟和血小板释放的生理位点。该项目的预期结果是阐明可以操纵以增加hIPSC血小板生产效率的特定生理信号传导途径。