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（hESCs）和诱导多能干细胞（iPS）在内的多能干细胞是独一无二的，因为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中分化细胞的发展可以为患者治疗提供无限的细胞供应。然而，令人惊讶的是，人们对如何控制这两种多能细胞的生长和分化知之甚少。特别是hESCs解离后的存活率小于1%。这限制了进行关键实验的能力，包括基因靶向，在无喂食器条件下分离均匀细胞群，以及开发大规模分化方案，这些都可能需要在提供患者量身定制的治疗之前进行。为了更好地了解如何提高hESCs的存活率，我们在细胞培养物中添加了个体生长因子来检查存活率。然而，这不是成本效益、数量或全面的。因此，在本提案中，我们将开发一种高含量筛选（HCS）系统来检测小分子提高生存率的潜力。我们将开发OCT4- egfp报告系，因为OCT4标记多能干细胞并在分化时迅速下调，以及开发无饲养器HCS检测，以跟踪特定培养系统中干细胞数量的变化。该提案的后半部分将致力于验证在HCS筛选中发现的潜在靶点，使用基于分子和细胞的分析来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，可以为未来的应用提供一个优秀的系统，用于监测来自患者特异性疾病模型的多种细胞系，遗传背景和干细胞系。

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.