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（由申请人提供）：在小鼠胚胎干细胞（mESC）被分离近20年后，1998年首次报道了人胚胎干细胞（hESC）的衍生，催生了hESC研究领域。虽然这一领域仅处于起步阶段，但hESC已经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许多不同细胞类型的显着潜力（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，用于治疗退行性、恶性或遗传性疾病，或由于炎症、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，hESC是研究人类正常和异常发育的宝贵研究工具，并可作为开发和测试新药的平台。 我们的长期目标是确定新的条件和分子程序，管理hESC的命运决定。如果我们最终将这些细胞用于治疗，这些知识是必不可少的。目前对hESC长期自我更新或谱系特异性分化的理解是非常初步的。为了解决这些问题，我们筛选了一小部分药理学抑制剂，并确定了一种蛋白质Ser-Thr激酶，作为控制hESC未分化生长的关键调控分子。该试验性研究为将大规模文库筛选应用于人胚胎干细胞的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，用于多能性或定向分化。这项研究的结果将为大规模的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，我们期望这些检测方法将为科学界研究hESC命运决定的分子机制提供新的工具，并可能有助于组织修复和再生的有效策略。人类胚胎干细胞（hESC）在了解人类早期发育以及发现和测试用于多种疾病的新药方面具有相当大的前景，包括心血管疾病，神经退行性疾病和糖尿病。为了实现人胚胎干细胞的治疗潜力，我们目前的实验建立高通量测定，以确定如何在分子水平上，这些细胞在培养中自我更新，并分化成体内特定类型的细胞。这些试验还可以促进产生足够的分化细胞，使我们能够评估hESC在临床前疾病模型中的治疗潜力，并为药物开发提供平台。