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

描述（由申请人提供）：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。衰竭心脏的收缩功能可以通过向心脏注射胚胎干细胞衍生的心肌细胞（ESCMs）来恢复，这些细胞整合到宿主组织中。为了促进ESCM的产生，需要可以量化ESCM产生的高通量筛选系统。拟议的研究将开发一种仪器，该仪器将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的ESCMs的发生。该系统将记录ESCM培养物的细胞内钙瞬变，这是分化心肌细胞的标志。该系统将以电极和控制器组件为特征，这些组件与商业高内涵显微镜工作站（例如，Beckman IC 100和阿默舍姆Incell 1000），并将基于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，以量化心脏特异性结构（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ESCMs分化的能力，并为进行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产生心脏细胞。以这种方式产生的心脏细胞可能用于治疗心脏病。

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.