Small Molecule Toolbox: Cardiomyocytes from Human Stem Cells

描述（由申请人提供）：据估计，超过500万美国人患有心力衰竭。2009年，美国社会因心脏病而付出的经济成本接近400亿美元。目前，晚期心力衰竭患者只有两种治疗选择，心脏移植或机械辅助心脏装置植入，但在美国，每年只有2,000多个心脏可供移植。与心肌衰竭或心肌衰弱有关的心脏病可以用药物或设备治疗，如心脏起搏器，心脏起搏器或植入泵。然而，在心脏病发作时，当心肌细胞死亡时，移植成为唯一的选择，因为人类心脏中的心肌细胞再生通常非常有限。目前使用的或临床试验中的方法不是为了再生心肌，而是依赖于改善剩余的心脏功能。在临床试验中的干细胞（SC）疗法的情况下，有益的效果是由于来自移植细胞的旁分泌信号或作为血管内皮细胞的持久性。由于大量的心脏病患者群体，开发心肌细胞替代疗法的努力正在进行中。在生物技术意义上生产心肌细胞是一个非常重要的目标，在药物发现和心力衰竭治疗方面都有相当大的应用。然而，尽管取得了进展，但提高干细胞或祖细胞成为人类心肌细胞的效率一直非常具有挑战性。提高心肌细胞产量的主要问题是缺乏有效的方法来诱导ESCs提供参与心脏发生的心肌细胞。一个关键问题是体外分化过程中心肌细胞的产量低。最重要的是需要以有效和具有成本效益的方式产生模拟心脏细胞的反应和生理行为的人类细胞。在大规模生物技术上将SC分化为心脏细胞的能力将导致几项进展。第一，大量细胞的技术将可用于移植目的。其次，CRO和大型制药公司将有大量的细胞可用于药物安全性评估。需要一种经济上可行的生物技术方法，使用容易获得和廉价的分化剂。在此，我们建议使用高含量和高通量细胞测定和动态药物化学的强大组合来开发纯的、易于制造的小分子“工具箱”化合物，以促进将分化成心肌细胞的hESC的诱导。有前途的心肌细胞分化剂（即，化合物1-3）已经被鉴定，并且这些试剂的改进和开发是本建议的重点。具体目标包括：1）在经验证的人ESC测定中测试740种结构上与1-3相关的化合物作为心肌细胞的诱导剂，和2）在经验证的反筛选中测试目标1的测试化合物以测试心肌细胞分化的选择性和作用模式。所提出的工作的成功完成将提供一个廉价的试剂工具箱，用于从人类胚胎干细胞诱导心肌细胞的广泛应用。 公共卫生相关性：在未来，人类干细胞疗法将为心脏病患者提供一种再生受损心肌细胞的方法。目前的治疗只能改善心脏功能，需要的是新的心肌细胞的产生。我们工作的目标是利用化学生物学来开发小分子“工具箱”化合物，这些化合物将刺激干细胞分化并产生人类心肌细胞。最终，这项工作的结果将提供工具箱试剂，用于培养心肌细胞，用于治疗心脏病的生物技术过程，并提高开发中人类药物的安全性。

DESCRIPTION (provided by applicant): It is estimated that more than 5 million Americans suffer from heart failure. The economic cost to US society for heart disease was almost $40 billion per in the year 2009. Currently, people with late-stage heart failure have only two treatment options, a heart transplant or implantation of a mechanically-assisted heart device but in the US, just over 2,000 hearts/year are available for transplantation. Heart diseases related to heart muscle failure or heart muscle weakening are treatable with drugs or devices such as defibrillators, pacemakers or implanted pumps. However, in heart attacks, when heart muscle cells die, transplantation becomes the only option because cardiomyocyte regeneration in the human heart is generally very limited. Current approaches used or in clinical trials are not designed to regenerate heart muscle but rely on improving remaining heart function. In the case of stem cell (SC) therapies in clinical trials, beneficial effects are due to paracrine signals from transplanted cells or persistence as vascular endothelial cells. Because of the large heart disease patient population, an intense effort to develop myocardial cell replacement therapies is underway. Production of cardiomyocytes in a biotech sense is a very important goal that would have considerable applications in both drug discovery and heart failure treatment. However, despite progress, increasing the efficiency of stem or progenitor cells to become human cardiomyocytes has been very challenging. The main problem with increasing the yield of cardiomyocytes is the lack of effective ways to induce ESCs to afford cardiomyocytes involved in cardiogenesis. A critical issue is the low yields of cardiomyocytes from in vitro differentiation processes. The need is to produce human cells that mimic the cardiac cell's response and physiological behavior in an efficient and cost-effective manner is paramount. The ability to differentiate SCs into cardiac cells on a large biotech scale will lead to several advances. First, technology for large quantities of cells will be available for transplantation purposes. Second, CROs and Big Pharma will have large numbers of cells available for drug safety evaluation. An economically viable biotechnological process using readily available and inexpensive differentiation agents is needed. Herein, we propose to use a powerful combination of high content and high throughput cellular assays and dynamic medicinal chemistry to develop pure, easy to make, small molecule "toolbox" compounds to promote the induction of hESCs that will differentiate into cardiomyocytes. Promising cardiomyocyte differentiation agents (i.e., compounds 1-3) have been identified and refinement and development of these agents is the focus of this proposal. The Specific Aims include: 1) Test 740 structurally related compounds to 1-3 as inducers of cardiomyocytes in a validated human ESC assay and 2) Test compounds of Aim 1 in validated counterscreens to test for selectivity and mode of action of cardiomyocyte differentiation. Successful completion of the proposed work will provide an inexpensive toolbox of reagents useful for the induction of cardiomyocytes from human ESCs of widespread utility. PUBLIC HEALTH RELEVANCE: In the future, human stem cell therapy will provide a way to regenerate damaged heart muscle cells for heart attack victims. Current therapies only improve heart function and what is needed is the generation of new heart muscle cells. The goal of our work is to use chemical biology to develop small molecule "toolbox" compounds that will stimulate stem cell differentiation and produce human cardiomyocytes. Ultimately, the results from this work will provide toolbox reagents for use to grow cardiomyocytes for use in a biotechnology process to treat heart disease and to improve the safety of human drugs in development.