MEGAKARYOCYTE REAGENTS SPECIFIC FOR THEIR MATURATION

本提案的总体目标是开发支持人巨核细胞体外发育的临床级巨核细胞发育培养基（MDM）。 使用这种独特的培养基将提供关于这些难以捉摸但至关重要的细胞的几个关键空白区域的信息;脂质代谢，成熟调节和临床应用。 其创新点在于增强脂质代谢的研究、细胞因子/基质分子组合的使用以及体内巨核细胞移植模型。 MDM将在开发血小板减少症和其他血小板疾病的疗法中具有临床用途，因为它允许产生大量的巨核细胞（> 10/9），这是通过常规骨髓抽吸不可能的。 MDM还允许实验室之间巨核细胞培养条件的标准化。 基于我们第一阶段研究的成功结果，我们在第二阶段提案中设计了相关和高度关键的实验，以研究以下目标。 具体目标1涉及MDM的进一步优化，特别关注增强巨核细胞脂质代谢。 具体目标2检查特定细胞因子和基质分子作为复杂调节信号的作用。 具体目标3是测试和/或长期植入。 该目的还为血小板疾病的基于细胞的疗法提供了临床前动物模型。 具体目标4是将这些培养物扩大到每个培养物产生1-5 x 10/9巨核细胞的水平。 该II期提案的成功完成将导致广泛使用的商业产品，临床相关的动物模型和急需的信息，以进一步研究这些生物学重要细胞的开发。 拟定商业应用： 支持巨核细胞增殖和分化的临床级试剂的开发将在开发用于治疗造血系统疾病如血小板减少症的新方法中是有价值的。

The overall objective of this proposal is to develop a clinical-grade Megakaryocyte Development Medium (MDM) that supports the ex vivo development of human megakaryocytes. Use of this unique medium will provide information on several critical gap-areas concerning these elusive yet critically important cells; lipid metabolism, regulation of maturation, and clinical utility. The innovative aspects are the studies on enhancing lipid metabolism, the use of cytokine/matrix molecule combinations, and an in vivo megakaryocyte-transplantation model. MDM will have clinical use in developing therapies for thrombocytopenia and other platelet disorders, in that it allows generation of large numbers of megakaryocytes (> 10/9) that are not possible via routine bone marrow aspiration. MDM also allows for the standardization of megakaryocyte culture conditions between laboratories. Building on the successful results of our Phase I studies, we have designed relevant and highly critical experiments in our Phase II proposal to investigate the following goals. Specific Aim 1 addresses the further optimization of MDM, with a particular focus on enhancing megakaryocyte lipid metabolism. Specific Aim 2 examines the effects of specific cytokines and matrix molecules as complex regulatory signals. Specific Aim 3 is to test and/or long-term engraftment. This aim also provides a pre-clinical animal model for cellular-based therapies of platelet disorders. Specific Aim 4 is to scale these cultures up to levels producing 1-5 x 10/9 megakaryocytes per culture. Successful completion of this Phase II proposal will result in a widely used commercial product, a clinically relevant animal model, and much needed information to further our studies on the development of these biologically important cells. PROPOSED COMMERCIAL APPLICATION: The development of clinical grade reagents that will support megakaryocyte proliferation and differentiation will be valuable in developing novel approaches for the treatment of hematopoietic disorders such as thrombocytopenia.