Regulating human pluripotent stem cell differentiation by colony confinement

描述（由申请人提供）：人多能干细胞，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，这两种特性赋予了用于产生用于发育研究、毒性测试和细胞治疗的正常人体细胞的强大系统。心肌细胞是一种特别有前途的细胞类型，可以从人多能干细胞衍生，因为心肌细胞不能容易地从初级来源或成体干细胞获得，并且在疾病和药物评价中具有巨大的重要性。虽然在从人多能干细胞产生收缩性心肌细胞方面已经取得了实质性进展，但产量仍然很低，并且细胞群是异质的，包含心房、心室和结心肌细胞，这是由于我们缺乏对支配心肌细胞发育的因素的基本理解。目前的分化方案利用化学因子来刺激多能干细胞的心脏发生。在最初的项目期间，我们确定细胞-细胞接触是从人胚胎干细胞中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/β-连环蛋白途径的信号传导减少相关，Want/β-连环蛋白途径对体内心肌细胞发育具有刺激和抑制作用。在下一个项目期间，我们建议阐明细胞间相互作用影响多能干细胞心肌细胞产量和类型特化的机制。具体而言，我们将测试以下假设：在分化期间将人多能干细胞限制在微孔中会影响通过调节由直接细胞-细胞接触和可溶性因子调节的Want/β-连环蛋白信号传导途径产生的心肌细胞的产量和类型。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集落形态和呈现调节Want/ -连环蛋白信号的生化信号线索的培养系统，并定量评估对心肌细胞发育的影响。我们检验这一假设的具体目的是：1。量化在胚状体形成期间人多能干细胞集落限制和定向分化为心肌细胞对所得心肌细胞的产量和表型的影响。 2.评价Want/β-连环蛋白信号传导在微孔限制的人多能干细胞中指定心肌细胞分化的机制作用。 3.评估用Want信号传导调节剂功能化的微孔控制人多能干细胞分化为心肌细胞的能力。

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.