Redirecting hemoglobin expression during Human ES Cell differentiation

描述（由申请人提供）：本申请涉及广泛挑战领域（14）干细胞和特定挑战主题14-DK-104：人胚胎干细胞（ES）/诱导多能干细胞（iPS）体外分化为NIDDK相关细胞/组织。 血红蛋白病，如镰状细胞病和地中海贫血，是不同强度的贫血，继续对美国劳动力中特定亚群的健康产生有害影响，最终对我们的经济生产力产生负面影响。我们有兴趣开发新的方法来改变正常的血红蛋白转换机制，使血红蛋白病患者可能受益于胎儿或胚胎珠蛋白表达的去抑制。简而言之，本发明中描述的想法涉及使用人胚胎干（ES）细胞，其被工程化以从其胎儿和成人珠蛋白启动子表达可检测的报告基因，以筛选导致更有效地诱导成人珠蛋白表达或再激活胎儿珠蛋白表达的小分子。这种方法的主要优点是，它避免了历史上使用小鼠来解决什么是人类基因调控模式，并且它利用了在ES细胞分化期间观察到的正常造血过程，这是一个不能在人类白血病细胞系中重现的过程。因此，本申请提出通过三个相互关联的目的来定义和利用改变正常血红蛋白转换机制的新方法：1）建立正确重现正常类球蛋白基因发育序列的有效hES细胞分化条件; 2）产生模拟该内源表达模式的正确调节的报告基因hES细胞系; 3）使用这条线来筛选小分子文库中改变/重定向类珠蛋白表达的化学物质。这种设计的一个关键方面是，目标3中的化学筛选，以及因此该提案的最终成功，不仅仅取决于目标1中成年产生细胞的建立。这非常适合14-DK-104类别，因为它旨在鉴定在分化的hES细胞中重定向血红蛋白表达的小分子，并且与NIDDK的计划利益一致。 公共卫生相关性：该项目的目标是建立一个标记的分子和细胞基线。在人类胚胎干细胞中，类似珠蛋白基因转换，然后将提供一个严格的和直接相关的途径，用于测试成人珠蛋白的诱导剂或胎儿珠蛋白表达的再激活剂。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells, and specific Challenge Topic 14-DK-104: In vitro differentiation of human Embryonic Stem Cells (ES)/Induced Pluripotent Stem Cells (iPS) to NIDDK relevant cells/tissues. Hemoglobinapathies, such as sickle cell disease and ¿-thalassemia, are anemias of varying intensity that continue to have a deleterious impact on the health of specific subpopulations in the US workforce, ultimately exerting a negative effect on our economic productivity. We have an interest in developing novel approaches to alter the normal hemoglobin switching mechanism so that patients with hemoglobinapathies might benefit from derepression of fetal or embryonic globin expression. Briefly, the idea described in the present proposal involves using human embryonic stem (ES) cells, engineered to express detectable reporters from their fetal and adult globin promoters, to screen for small molecules that lead to more efficient induction of adult globin, or reactivation of fetal globin, expression. The major advantages of this approach is that it avoids the historical use of the mouse to address what is a human gene regulatory pattern, and that it takes advantage of the normal hematopoietic process observed during ES cell differentiation, a process that cannot be recapitulated within human leukemic cell lines. As a result, this application proposes to define and utilize a novel approach to alter the normal hemoglobin switching mechanism via three interrelated aims: 1) establish efficient hES cell differentiation conditions that correctly recapitulate the normal ¿-like globin gene developmental sequence; 2) generate a correctly regulated reporter hES cell line that mimics this endogenous expression pattern; 3) use this line to screen a small molecule library for chemicals that alter/redirect ¿-like globin expression. A key aspect of this design is that the chemical screen in Aim 3, and thus the ultimate success of the proposal, is not solely dependent on establishment of adult ¿-producing cells in Aim 1. This fits well into the 14-DK-104 category, as it aims to identify small molecules that redirect hemoglobin expression in differentiating hES cells and is consistent with programmatic interests of the NIDDK. PUBLIC HEALTH RELEVANCE: The goals of this project are to establish a molecular and cellular baseline of marked ¿.like globin gene switching in human embryonic stem cells that will then provide a rigorous and directly relevant avenue for testing inducers of adult globin or reactivators of fetal globin expression.