Controlling an Ontogenic Masterswitch to Maximize Thrombopoiesis

控制个体发生主开关以最大化血小板生成

• 批准号: 9142354
• 负责人: Adam N. Goldfarb
• 金额: $ 44.59万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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/9142354
• 关键词: Acute Megakaryocytic Leukemias; Adult; Affect; Agonist; Binding; Bioreactors; Blood Platelets; Bone Marrow; Cells; Clinical; Collection; Complex; Data; Defect; Development; Dissection; Down Syndrome; Down-Regulation; Ectopic Expression; Engineering; Environment; Equilibrium; Goals; Growth; H19 gene; HMGA2 gene; Health; Hemostatic function; Human; Immunization; Immunodeficient Mouse; Life; Maps; Megakaryocyte Proliferation; Megakaryocytes; Megakaryocytopoieses; Modeling; Molecular; Morphogenesis; Neonatal; Pathogenesis; Pathway interactions; Patients; Perfusion; Phenotype; Phosphotransferases; Platelet Count measurement; Platelet Transfusion; Pluripotent Stem Cells; Positive Transcriptional Elongation Factor B; Production; Protein Isoforms; Risk; Signal Pathway; Signal Transduction; Site; Somatic Cell; Staging; Stem cells; System; Therapeutic; Thrombopoiesis; Transcriptional Activation; Translations; Untranslated RNA; analog; base; cost effective; fetal; human embryonic stem cell; knock-down; neonatal human; novel; paralogous gene; progenitor; programs; research study; scale up; self-renewal; small hairpin RNA; transmission process

 DESCRIPTION (provided by applicant): A pressing need exists for clinically applicable systems for ex vivo platelet production. Multiple technological advances have set the stage to attain this goal. Inducible pluripotent stem cells (iPSC) derived from donor somatic cells can now be manipulated to yield customized, expandable megakaryocyte (Mk) progenitors. Perfusion bioreactors that replicate the bone marrow environment have enhanced the efficiency of functional platelet release and collection. The biggest roadblock in clinical translation consiss of the problem of scalability. In particular, highly proliferative Mk progenitors yield poor platelt numbers, and Mk with high platelet yields come from progenitors with very limited prolferative capacity. An ability to circumvent these limitations by combining progenitor expandability with efficient platelet production will be critical for cost- effective scale-up. The efficiency of platlet production depends on a program of Mk morphogenesis involving massive cellular enlargement and polyploidization. The relative balance of morphogenetic versus proliferative potential depends on ontogenic developmental stage. Thus, fetal and neonatal Mk progenitors show extensive self-renewal but limited morphogenesis. Progenitors derived from human ESC and iPSC recapitulate the features of early fetal megakaryopoiesis: high proliferation with minimal morphogenesis. The influence of ontogenic stage affects not only platelet numbers but also extends to platelet function. Specifically, Mk from earlier in ontogeny yield platelets with proportionally diminished aggregation capacity. Our lab identified a signaling pathway critical in Mk morphogenesis (Elagib et al. Dev. Cell, 2013). In this pathway, high-amplitude activation of the transcriptional kinase P-TEFb occurs due to downregulation of the noncoding RNA 7SK. In new unpublished data, we find that defects in this Mk morphogenesis pathway underlie the phenotypic differences between neonatal and adult Mk. Specifically, neonatal Mk fail to downregulate 7SK and fail to trigger high-amplitude activation of P-TEFb. We have identified a 7SK binding factor, IGF2BP3, that is present only in neonatal Mk and functions as an ontogenic masterswitch in Mk morphogenesis. Antagonism of IGF2BP3 by either shRNA knockdown or a novel inhibitory compound significantly augments morphogenesis in neonatal Mk. Conversely, ectopic IGF2BP3 converts adult MK into a fetal phenotype, and a putative agonist compound augments fetal-like features in neonatal Mk. IGF2BP3 thus represents a highly attractive target for engineering scalable megakaryopoiesis. In human neonatal Mk, it appears to be the key determinant of Mk ontogenic phenotype. From a therapeutic perspective, it is a "druggable" target, with the capability of both negative and positive modulation. Aim 1 will examine the morphogenesis signaling pathway in iPSC Mk, the contributions of IGF2BP factors, and optimal approaches to enhance thrombopoiesis. Aim 2 will take a complementary approach and determine circuits necessary for fetal reprogramming of adult Mk progenitors, thereby allowing for their large-scale, reversible expansion.

 描述（由申请人提供）：迫切需要用于体外血小板生产的临床适用系统。多项技术进步为实现这一目标奠定了基础。从供体体细胞衍生的诱导性多能干细胞（iPSC）现在可以被操纵以产生定制的、可扩增的巨核细胞（Mk）祖细胞。复制骨髓环境的灌注生物反应器提高了功能性血小板释放和收集的效率。临床翻译的最大障碍是可扩展性问题。特别是，高度增殖的Mk祖细胞产生的血小板数量少，而具有高血小板产量的Mk来自增殖能力非常有限的祖细胞。通过将祖细胞可扩增性与有效的血小板产生相结合来规避这些限制的能力对于成本有效的规模扩大将是至关重要的。血小板生产的效率取决于Mk形态发生的程序，包括大量的细胞扩大和多倍化。形态发生与增殖潜力的相对平衡取决于个体发育阶段。因此，胎儿和新生儿Mk祖细胞表现出广泛的自我更新，但有限的形态发生。来源于人ESC和iPSC的祖细胞概括了早期胎儿巨核细胞生成的特征：高增殖和最小的形态发生。个体发育阶段的影响不仅影响血小板数量，而且还延伸到血小板功能。具体地，来自个体发育早期的Mk产生具有成比例减少的聚集能力的血小板。 我们的实验室发现了一种信号通路， Mk形态发生（Elagib等人，Dev. Cell，2013）。在该途径中，由于非编码RNA 7SK的下调，转录激酶P-TEFb发生高幅度激活。在新的未发表的数据中，我们发现，在这个MK形态发生途径的缺陷的基础新生儿和成人MK之间的表型差异。具体而言，新生儿Mk未能下调7SK，并且未能触发P-TEFb的高振幅激活。我们已经确定了一个7SK结合因子，IGF 2BP 3，这是目前只在新生儿Mk和功能作为一个个体发育的主开关Mk形态发生。通过shRNA敲低或新型抑制性化合物对IGF 2BP 3的拮抗作用显著增强新生Mk的形态发生。相反，异位IGF 2BP 3将成人MK转化为胎儿表型，并且推定的激动剂化合物增强新生儿MK中的胎儿样特征。IGF 2BP 3因此代表了用于工程化可扩展的巨核细胞生成的高度有吸引力的靶标。在人类新生儿Mk中，它似乎是Mk个体发生表型的关键决定因素。从治疗的角度来看，它是一个“可药物化”的目标，具有负调节和正调节的能力。目的1将研究iPSC Mk中的形态发生信号通路，IGF 2BP因子的贡献，以及增强血小板生成的最佳方法。目标2将采取一种互补的方法，并确定成年Mk祖细胞的胎儿重编程所需的电路，从而使其大规模，可逆的扩张。