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)：迫切需要临床上可用于体外血小板生产的系统。多种技术进步为实现这一目标奠定了基础。来自供体细胞的可诱导多能干细胞(IPSC)现在可以被操纵来产生定制的、可扩增的巨核细胞(Mk)祖细胞。复制骨髓环境的灌流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，高增殖的Mk祖细胞产生的血小板数量很少，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。通过将祖细胞的扩张性与高效的血小板生产相结合来绕过这些限制的能力将是具有成本效益的放大的关键。血小板生产的效率依赖于一种包括大量细胞放大和多倍化的Mk形态发生程序。形态发生和增殖潜能的相对平衡取决于个体发育阶段。因此，胎儿和新生儿的MK祖细胞表现出广泛的自我更新，但形态发生有限。来源于人类ESC和IPSC的祖细胞概括了早期胎儿巨核生成的特征：高增殖和最少的形态发生。个体发生阶段的影响不仅影响到血小板数量，还影响到血小板的功能。具体地说，个体发育早期的Mk产生的血小板聚集能力成比例地降低。我们的实验室发现了一条关键的信号通路 MK形态发生(Elagib等人戴夫。CELL，2013)。在这一途径中，由于非编码RNA 7SK的下调，转录激酶P-TEFb发生了高幅度的激活。在新的未发表的数据中，我们发现这种Mk形态发生途径的缺陷是新生儿和成人Mk表型差异的基础。具体地说，新生儿Mk不能下调7SK，也不能触发P-TEFb的高幅度激活。我们已经确定了一个7SK结合因子IGF2BP3，它只存在于新生儿Mk中，在Mk的形态发生中起着个体发生主开关的作用。通过shRNA敲除或一种新的抑制化合物拮抗IGF2BP3显著增强新生儿Mk的形态发生。相反，异位IGF2BP3将成人MK转化为胎儿表型，而一种假定的激动剂化合物增强了新生儿MK的胎儿样特征。因此，IGF2BP3是工程化可伸缩巨核细胞的一个极具吸引力的靶点。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“可用药”的靶点，既有负向调节能力，也有正向调节能力。目的1研究IPSC Mk的形态发生信号通路，IGF2BP因子的作用，以及增强血小板生成的最佳途径。AIM 2将采取补充方法，确定成年MK祖细胞胎儿重编程所需的电路，从而允许它们大规模、可逆地扩张。