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: 9276795
• 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/9276795
• 关键词: Acute Megakaryocytic Leukemias; Adult; Affect; Agonist; Binding; Bioreactors; Blood Platelets; Bone Marrow; Cells; Collection; Complex; Critical Pathways; Custom; Data; Defect; Development; Dissection; Down Syndrome; Down-Regulation; Ectopic Expression; Engineering; Environment; Equilibrium; Goals; Growth; H19 gene; HMGA2 gene; Hemostatic function; Human; Immunization; Immunodeficient Mouse; Life; Megakaryocyte Proliferation; Megakaryocytes; Megakaryocytopoieses; Modeling; Molecular; Morphogenesis; Neonatal; Pathogenesis; Pathway interactions; Patients; Perfusion; Pharmacology; 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; Stem cells; System; Therapeutic; Thrombopoiesis; Transcriptional Activation; Untranslated RNA; analog; clinical application; clinical translation; cost effective; experimental study; fetal; human embryonic stem cell; knock-down; neonatal human; novel; paralogous gene; progenitor; programs; public health relevance; scaffold; 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）：迫切需要用于离体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。现在可以操纵源自供体体细胞的诱导多能干细胞 (iPSC) 来产生定制的、可扩展的巨核细胞 (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较差，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。通过将祖细胞扩增性与高效血小板生产相结合来规避这些限制的能力对于具有成本效益的规模化至关重要。血小板产生的效率取决于涉及大量细胞增大和多倍体化的 Mk 形态发生程序。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，但形态发生有限。来自人类 ESC 和 iPSC 的祖细胞概括了早期胎儿巨核细胞生成的特征：高度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，还影响血小板功能。具体来说，个体发育早期的 Mk 产生的血小板聚集能力成比例降低。 我们的实验室发现了一条至关重要的信号通路 Mk 形态发生（Elagib 等人 Dev. Cell，2013）。在此通路中，由于非编码 RNA 7SK 的下调，转录激酶 P-TEFb 发生大幅激活。在新的未发表的数据中，我们发现 Mk 形态发生途径的缺陷是新生儿和成人 Mk 之间表型差异的基础。具体来说，新生儿 Mk 无法下调 7SK，也无法触发 P-TEFb 的高幅度激活。我们已经鉴定出一种 7SK 结合因子 IGF2BP3，它仅存在于新生儿 Mk 中，并在 Mk 形态发生中充当个体发育主开关。通过 shRNA 敲低或新型抑制化合物对 IGF2BP3 的拮抗作用可显着增强新生儿 Mk 的形态发生。相反，异位 IGF2BP3 将成人 MK 转化为胎儿表型，而推定的激动剂化合物增强了新生儿 Mk 的胎儿样特征。因此，IGF2BP3 代表了工程可扩展巨核细胞生成的一个极具吸引力的靶标。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“可成药”的靶点，具有负调节和正调节的能力。目标 1 将检查 iPSC Mk 中的形态发生信号通路、IGF2BP 因子的贡献以及增强血小板生成的最佳方法。目标 2 将采取补充方法，确定胎儿对成人 Mk 祖细胞进行重编程所需的电路，从而实现大规模、可逆的扩增。