Cell-based high-throughput assays for fate decisions of human embryonic stem cell

基于细胞的高通量测定人类胚胎干细胞的命运决定

• 批准号: 7676097
• 负责人: Fei Wang
• 金额: $ 28.22万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676097
• 关键词: Address; Biological Assay; Cardiac Myocytes; Cardiovascular Diseases; Cell Line; Cell Therapy; Cells; Collection; Communities; Derivation procedure; Diabetes Mellitus; Disease; Ectoderm; Endoderm; Ensure; Fluorescence; Goals; Growth; Heart failure; Hereditary Disease; Human Development; Infection; Inflammation; Injury; Knowledge; Libraries; Luciferases; Malignant - descriptor; Mesoderm; Molecular; Mus; Natural regeneration; Nerve Degeneration; Neurons; Pancytopenia; Parkinson Disease; Pharmaceutical Preparations; Phosphotransferases; Pilot Projects; Pre-Clinical Model; Process; Production; Proteins; Reporter; Reporter Genes; Reporting; Research; Screening procedure; Source; Spinal cord injury; Staging; Stimulus; Structure of beta Cell of islet; Testing; Therapeutic; Trauma; Undifferentiated; Wound Healing; base; cell type; drug development; embryonic stem cell; high throughput screening; human embryonic stem cell; infancy; inhibitor/antagonist; mTOR protein; pluripotency; precursor cell; programs; promoter; research study; self renewing cell; self-renewal; tool

DESCRIPTION (provided by applicant): Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESCs) in 1998 spawned the field of hESC research. Although this field is only in its infancy, hESCs have already been shown to be capable of long-term self-renewal in culture and have remarkable potential to develop into many different cell types in the body (known as pluripotency). They therefore represent a theoretically inexhaustible source of precursor cells to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. Meanwhile, hESCs are an invaluable research tool to study human development, both normal and abnormal, and can serve as a platform to develop and test new drugs. Our long-term goal is to define new conditions and molecular programs that govern fate decisions of hESCs. The knowledge is essential if we are ultimately to use these cells for therapy. The current understanding of hESC long-term self-renewal or lineage-specific differentiation is extremely rudimentary. As an attempt to address these questions, we screened a small collection of pharmacological inhibitors and identified a protein Ser-Thr kinase, as a key regulatory molecule that controls the undifferentiated growth of hESCs. This pilot study provided proof-of-concept for applying large-scale library screening to the study of hESCs. Accordingly, we will develop cell-based high-throughput assays by establishing hESCs containing enhanced green fluorescence protein (EGFP) or luciferase reporters for pluripotentcy or for directed differentiation. The results from the study will set the stage for large-scale library-screening efforts for searching molecules that influence the fate decisions of hESCs. Therefore, we expect that these assays will provide new tools for the scientific community to study the molecular mechanisms underlying hESC fate determination and may contribute to effective strategies for tissue repair and regeneration. Human embryonic stem cells (hESCs) hold considerable promise for understanding early human development and for finding and testing new drugs for a vast number of conditions, including cardiovascular diseases, neurodegenerative processes and diabetes. To realize the therapeutic potential of hESCs, we present experiments to establish high throughput assays for determining how, at the molecular level, these cells self-renew in culture and differentiate into a specific type of cells in the body. These assays may also facilitate production of sufficient differentiated cells to allow us to assess the therapeutic potential of hESCs in preclinical models of diseases, and to offer platforms for drug development.

描述（申请人提供）：在鼠胚胎干细胞（mESC）被分离近20年后，1998年第一份关于人胚胎干细胞（hESC）衍生的报告催生了hESC研究领域。尽管这一领域还处于起步阶段，但 hESC 已被证明能够在培养中长期自我更新，并且具有在体内发育成多种不同细胞类型的巨大潜力（称为多能性）。因此，它们代表了理论上取之不尽的前体细胞来源，可用于治疗退行性、恶性或遗传性疾病，或炎症、感染和创伤引起的损伤。这种多能细胞被誉为治疗糖尿病、帕金森病、阿尔茨海默病、脊髓损伤、心力衰竭和骨髓衰竭的可能手段。同时，hESCs是研究人类正常和异常发育的宝贵研究工具，并且可以作为开发和测试新药的平台。 我们的长期目标是定义控制 hESC 命运决定的新条件和分子程序。如果我们最终要使用这些细胞进行治疗，这些知识至关重要。目前对 hESC 长期自我更新或谱系特异性分化的理解还非常初级。为了解决这些问题，我们筛选了一小部分药理学抑制剂，并鉴定了一种蛋白质 Ser-Thr 激酶，作为控制 hESC 未分化生长的关键调节分子。这项试点研究为将大规模文库筛选应用于 hESCs 研究提供了概念验证。因此，我们将通过建立含有增强型绿色荧光蛋白（EGFP）或荧光素酶报告基因的 hESC 来开发基于细胞的高通量测定，以实现多能性或定向分化。该研究的结果将为大规模文库筛选工作奠定基础，以寻找影响 hESC 命运决定的分子。因此，我们期望这些检测将为科学界提供新的工具来研究 hESC 命运决定的分子机制，并可能有助于制定组织修复和再生的有效策略。人类胚胎干细胞（hESC）对于了解人类早期发育以及寻找和测试治疗多种疾病（包括心血管疾病、神经退行性过程和糖尿病）的新药具有广阔的前景。为了实现 hESC 的治疗潜力，我们进行了实验，建立高通量测定法，以确定这些细胞如何在分子水平上在培养物中自我更新并分化为体内特定类型的细胞。这些测定还可以促进足够分化细胞的产生，使我们能够评估 hESC 在疾病临床前模型中的治疗潜力，并为药物开发提供平台。