Live cell and HCS assays to quantify production of cardiomyocytes from stem cells

描述(申请人提供)：心力衰竭是现代社会死亡的主要原因，是心肌细胞死亡的结果。通过将胚胎干细胞来源的心肌细胞(ESCM)整合到宿主组织中，可以恢复衰竭心脏的收缩功能。为了促进ESCM的生产，需要能够量化ESCM生产的高通量筛查系统。这项拟议的研究将开发一种仪器，利用活细胞和高含量筛选(HCS)技术来量化在96孔培养皿中培养的干细胞发生ESCM的情况。该系统将记录ESCM培养的细胞内钙瞬变，这是分化的心肌细胞的标志。该系统将以连接商用高含量显微镜工作站(例如Beckman IC100和amersham Incell 1000)的电极和控制器组件为特色，并将根据荧光转基因的表达来区分细胞。还将开发用于自动图像分析的染色试剂和软件，以量化心脏特定结构(肌原纤维)和蛋白质(SERCA2)的细胞表达和组织。该系统将能够测试候选化学品影响ESCM分化的能力，并为开展分化过程的化学基因组研究提供平台。我们将开发一种仪器，帮助研究人员从胚胎干细胞中培育出心脏细胞。以这种方式产生的心脏细胞可能被用于治疗心脏病。

DESCRIPTION (provided by applicant): Heart failure is a leading cause of mortality in modern society, and is the result from the death of cardiac myocytes. Contractile function of failing hearts can be restored by injecting the heart with embryonic stem cell-derived cardiac myocytes (ESCMs) which integrate into the host tissue. To facilitate production of ESCMs, high throughput screening systems are needed that can quantify ESCM production. The proposed research will develop an instrument that will utilizes live cell and high content screening (HCS) techniques to quantify the occurrence of ESCMs for stem cells cultured in 96-well dishes. The system will record intracellular calcium transients from ESCM cultures, which are a hallmark of differentiated cardiac myocytes. The system will feature electrode and controller assemblies that interface to commercial high content microscopy workstations (e.g., the Beckman IC100 and the Amersham Incell 1000) and will discriminate cells based on expression of fluorescent transgenes. Staining reagents and software for automated image analysis will also be developed to quantify the cellular expression and organization of cardiac-specific structures (myofibrils) and proteins (SERCA2). The system will enable testing of candidate chemicals for their ability to influence differentiation of ESCMs and provide a platform for conducting chemical genomic studies of the differentiation process. We will develop an instrument that will help researchers produce heart cells from embryonic stem cells. Heart cells produced in this manner can potentially be used to treat heart disease.