Regulating human pluripotent stem cell differentiation by colony confinement

描述(申请人提供)：人类多能干细胞，包括胚胎干细胞和诱导多能干细胞，提供了无限自我更新潜力和多能性的独特组合，这两个特性为发育研究、毒性测试和细胞治疗提供了一个强大的系统来产生正常的人类体细胞。心肌细胞是一种特别有前景的细胞类型，可以从人类多能干细胞中获得，因为心肌细胞不容易从原代来源或成体干细胞中获得，在疾病和药物评价中具有极其重要的意义。尽管人类多能干细胞在培养收缩心肌细胞方面取得了实质性进展，但由于我们对控制心肌细胞发育的因素缺乏基本的了解，产量仍然很低，细胞群是不同的，包括心房、心室和结节心肌细胞。目前的分化方案利用化学因素来刺激多能干细胞的心脏生成。在最初的项目阶段，我们发现细胞间的接触是人类胚胎干细胞产生心肌细胞的重要因素，并发现存在产生心肌细胞的最佳集落大小。这种最佳大小与通过Want/-catenin途径的信号减少有关，该途径对活体心肌细胞的发育既有刺激作用，也有抑制作用。在下一个项目阶段，我们建议阐明细胞间相互作用影响从多能干细胞中获得心肌细胞的产量和类型指定的机制。具体地说，我们将测试这样一个假设，即在分化过程中将人类多能干细胞限制在微孔中，会影响通过调节Want/-catenin信号通路而产生的心肌细胞的产量和类型，该信号通路受细胞与细胞的直接接触和可溶性因子的调节。我们团队在生物材料开发、干细胞生物学、信号通路分析和心肌细胞生理学方面的专业知识将使我们能够构建系统地改变菌落形态和呈现调控Want/-catenin信号的生化信号信号的培养系统，并定量评估对心肌细胞发育的影响。我们的具体目的是验证这一建议的假设：1.量化人多能干细胞在拟胚体形成过程中的集落限制和定向分化为心肌细胞对所产生的心肌细胞产量和表型的影响。2.评价在微孔限制的人类多能干细胞中，Want/-catenin信号在确定心肌细胞分化中的机制作用。3.评价带有WANT信号调节因子的微孔调控人多能干细胞向心肌细胞分化的能力。 公共卫生相关性：人类多能干细胞来源的心肌细胞提供了一种研究人类心脏体外发育的系统，一种筛选治疗心脏疾病的药物有效性和评估其他药物的心脏毒性的工具，以及一种治疗受损和疾病心脏的潜在细胞疗法。了解细胞间的接触如何调节心肌细胞的分化将提高我们对人类心脏发育的理解，允许合理设计方法学来提高多潜能干细胞来源的心肌细胞的产量和功能，并促进多潜能干细胞来源的心肌细胞的翻译应用于研究和临床应用。

DESCRIPTION (provided by applicant): Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating normal human somatic cells for developmental studies, toxicity testing, and cellular therapies. Cardiac myocytes are a particularly promising cell type that can be derived from human pluripotent stem cells since cardiac myocytes cannot easily be attained from primary sources or adult stem cells and are of tremendous importance in disease and pharmaceutical evaluation. While substantial progress has been made in generating contractile cardiac myocytes from human pluripotent stem cells, yields remain low and cell populations are heterogeneous, containing atrial, ventricular, and nodal cardiac myocytes, as a result of our lack of fundamental understanding of factors that govern cardiac myocyte development. Current differentiation protocols utilize chemical factors to stimulate cardiogenesis in pluripotent stem cells. In the initial project period we identified cell-cell contact as an important contributor to cardiac myocyte yield from human embryonic stem cells and discovered that an optimum colony size exists for generating cardiac myocytes. This optimum size correlates with reduced signaling through the Want/ -catenin pathway, which has both stimulatory and inhibitory effects on cardiac myocyte development in vivo. In the next project period we propose to elucidate the mechanism by which intercellular interactions affect yield and type specification of cardiac myocytes from pluripotent stem cells. Specifically, we will test the hypothesis that confinement of human pluripotent stem cells in microwells during differentiation affects the yield and type of cardiac myocytes generated by modulating the Want/ -catenin signaling pathway which is regulated by direct cell-cell contact and soluble factors. Our team's expertise in biomaterials development, stem cell biology, signaling pathway analysis, and cardiac myocyte physiology will permit us to construct culture systems that systematically vary colony morphology and presentation of biochemical signaling cues that regulate Want/ -catenin signaling, and quantitatively assess the effects on cardiac myocyte development. Our specific aims to test the hypothesis of this proposal are: 1. quantify the effects of human pluripotent stem cell colony confinement during embryoid body formation and directed differentiation to cardiac myocytes on the yield and phenotype of the resulting cardiac myocytes. 2. Evaluate the mechanistic role of Want/ -catenin signaling in specifying cardiac myocyte differentiation in microwell-confined human pluripotent stem cells. 3. Assess the ability of microwells functionalized with Want signaling regulators to control human pluripotent stem cell differentiation to cardiac myocytes. PUBLIC HEALTH RELEVANCE: Cardiac myocytes derived from human pluripotent stem cells offer a system to study development of the human heart in vitro, a tool to screen the effectiveness of drugs to treat cardiac diseases and to assess cardiac toxicity of other pharmacologic agents, and a potential cellular therapy to treat damaged and diseased hearts. Understanding how cell-cell contact regulates cardiac myocyte differentiation will improve our understanding of human heart development, permit rational design of methodologies to improve yield and functionality of pluripotent stem cell-derived cardiac myocytes, and facilitate translational applications of pluripotent stem cell- derived cardiac myocytes to research and clinical applications.