Development of High Content Screens for Human Pluripotent Stem Cells

描述（由申请人提供）：包括人胚胎干细胞（hESC）和诱导多能干细胞（iPS）的多能干细胞的独特之处在于它们可以自我更新和/或分化为来自每个胚层的细胞。从hESC或iPS分化的细胞的发展可以提供无限的细胞供应用于患者治疗。然而，令人惊讶的是，关于如何控制这些多能细胞类型中的任一种的生长和分化所知甚少。特别地，解离后hESC的存活率小于1%。这限制了进行关键实验的能力，所述关键实验包括基因靶向、在无饲养层条件下分离同质细胞群以及开发大规模分化方案，在提供患者定制的疗法之前可能需要其中的每一个。为了更好地了解如何提高hESC的存活率，我们在细胞培养物中加入了单独的生长因子来检测存活率。然而，这并不具有成本效益，也不定量或全面。因此，在本提案中，我们将开发一种高含量筛选（HCS）系统，以测定小分子改善存活率的潜力。我们将开发OCT4-EGFP报告细胞系，因为OCT4标记多能干细胞并在分化后迅速下调，以及开发无饲养层HCS测定以跟踪确定的培养系统中干细胞数量的变化。该提案的第二部分将致力于使用基于分子和细胞的测定来验证HCS筛选中发现的潜在靶点，以跟踪小分子治疗对多能干细胞存活的影响。用于hESC或iPS的HCS的开发是新颖的，并且可以在未来的应用中提供用于监测来自患者特异性疾病模型的多个细胞谱系、遗传背景和干细胞系的优秀系统。 公共卫生相关性：了解多能干细胞如何做出生存或自我更新与分化的决定尚不清楚。在使用从干细胞获得的分化细胞之前，我们需要开发更好的检测方法来了解如何在培养中维持（或消除）这些细胞。控制这种决定的一种机制是调节干细胞在培养中的存活方式。因此，在本提案中，我们将开发一种高含量筛选（HCS）试验，该试验将允许检查多个参数，包括干细胞存活、细胞形态和大小以及多能性丧失。还将在确定的培养系统中开发测定;因此，小分子对干细胞存活的影响将直接影响培养物中的干细胞。在开发用于患者的细胞疗法之前，控制多能干细胞生长的能力是至关重要的，并且HCS的开发将提供用于在统一系统中评估多个干细胞系的关键工具。

DESCRIPTION (provided by applicant): Pluripotent stem cells including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPS) are unique in that they can self-renew and/or differentiate into cells from every germ layer. Development of differentiated cells from either hESC or iPS could provide an unlimited supply of cells for use in patient therapy. However, surprisingly little is known about how to control growth and differentiation of either of these pluripotent cell types. In particular, the survival rate of hESCs after dissociation is less than 1%. This limits the ability to perform critical experiments including gene targeting, isolation of homogeneous populations of cells in feeder free conditions, and development of large scale differentiation protocols, each of which may be required prior to providing patient tailored therapy. In order to develop a greater understanding of how to improve survival of hESCs, we have added individual growth factors to growing cultures of cells to examine survival. However, this is not cost-effective, quantitative or comprehensive. Therefore in this proposal we will develop a high content screening (HCS) system to assay the potential of small molecules to improve survival. We will develop an OCT4-EGFP reporter line as OCT4 marks pluripotent stem cells and gets rapidly down regulated upon differentiation, as well as development of feeder free HCS assays to follow changes in stem cell numbers in a defined culture system. The second half of this proposal will be dedicated to validating potential targets found in the HCS screens using both molecular and cellular based assays to follow the effects of small molecule treatment on pluripotent stem cell survival. Development of HCS for either hESC or iPS is novel and could provide an excellent system in future applications for monitoring multiple cell lineages, genetic backgrounds and stem cell lines from patient-specifc disease models. PUBLIC HEALTH RELEVANCE: Understanding how pluripotent stem cells make decisions to survive or self-renew versus differentiate is not well understood. Prior to using differentiated cells obtained from stem cells we will need to develop better assays to understand how to maintain (or eliminate) these cells in culture. One mechanism of controlling this decision is to regulate how stem cells survive in culture. Therefore in this proposal we will develop a high content screening (HCS) assay, which will allow for examination of multiple parameters including stem cell survival, cell morphology and size as well as loss of pluripotency. The assays will also be developed in defined culture systems; therefore the effects of small molecules on stem cell survival will be direct to the stem cells in culture. The ability to control growth of pluripotent stem cells is critical prior to development of cell therapy for patients and the development of HCS will provide a critical tool for evaluating multiple stem cell lines in a uniform system.