High throughput screening of embryonic stem cell differentiation

胚胎干细胞分化的高通量筛选

• 批准号: 7613572
• 负责人: Ngan F. Huang
• 金额: $ 5.17万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7613572
• 关键词: Affect; American; Biological; Biological Assay; Bioluminescence; Blood flow; Bone Marrow; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cell Separation; Cell physiology; Cells; Collagen; Collagen Type IV; Conditioned Culture Media; Data; Endothelial Cells; Endothelium; Extracellular Matrix; Fibronectins; Firefly Luciferases; Gangrene; Gelatin; Genes; Goals; Growth Factor; Hematopoietic stem cells; Hindlimb; Histologic; Image; Immunoassay; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Ischemia; Knowledge; Laminin; Lasers; Lead; Limb structure; Measurement; Mediating; Mesenchymal Stem Cells; Morbidity - disease rate; Morphology; Mus; Natural regeneration; PTPRC gene; Patients; Pericytes; Peripheral arterial disease; Phenotype; Pluripotent Stem Cells; Polymerase Chain Reaction; Process; Property; Proteins; Proto-Oncogene Protein c-kit; Public Health; Research; Screening procedure; Source; Spectrum Analysis; Spottings; Staining method; Stains; Stem cells; Symptoms; Technology; Teratoma; Time; Tube; Ulcer; Validation; Vascular Endothelial Cell; angiogenesis; cadherin 5; cell type; cytokine; embryonic stem cell; functional improvement; high throughput screening; improved; improved functioning; in vitro Assay; in vivo; in vivo Model; innovation; matrigel; migration; mortality; promoter; red fluorescent protein; research study; stem cell differentiation

DESCRIPTION (provided by applicant): Over 8 million Americans suffer from peripheral arterial disease (PAD), which is typically due to atherosclerotic occlusive disease of the peripheral arteries of the limbs, causing symptoms such as ischemic ulcerations or gangrene. Biological approaches to improve limb blood flow by regenerating the endothelium seem promising for treatment of PAD. A potential cell source for endothelial regeneration is embryonic stem cells (ESCs), which are pluripotent stem cells that can differentiate into endothelial cells (ECs). However, the ability to direct differentiation into ECs remains challenging due to the low yields (typically less than 5%). ESC differentiation is known to be affected by growth factors in the conditioned medium and by the matrix upon which they are grown. With respect to determining the optimal matrix composition on ESC differentiation, I propose to utilize extracellular matrix (ECM) microarrays. This is a high-throughput approach for screening several hundreds of ECMs and ECM mixtures of defined compositions for matrix- mediated ESC differentiation into EC lineage. Therefore, the goal of this project is optimize matrix conditions that favor ESC differentiation into ECs, and then characterize the phenotype and angiogenic properties of ESC-derived ECs in vitro and in vivo. First, I will characterize the differentiation of ESC-derived ECs in vitro using a high throughput ECM microarray approach. The microarray will be comprised of micro-scale spots containing single or mixtures of various matrix proteins. For easy purification and non-invasive tracking, I will utilize ESCs transduced with a lentiviral construct that consists of an EC-specific promoter, VE-cadherin, and contains genes encoding firefly luciferase for bioluminescence imaging and red fluorescent protein for immunofluorescent tracking. Next, I will differentiate ESCs on the ECM array and select the matrix composition that optimizes EC differentiation for large-scale differentiation experiments. I will then purify ECs by fluorescently activated cell sorting and characterize the cells for EC phenotypic morphology and function. Finally, using an in vivo model of PAD (hindlimb ischemia), I will determine whether ESC-derived ECs can survive in the ischemic limb, incorporate into the vasculature, and improve function by bioluminescence tracking and laser Doppler spectroscopy flow measurement. In summary, this project will utilize innovative strategies to enhance directed differentiation of ESCs into ECs for vascular regeneration for patients suffering from PAD. The potential impact of this research to public health is a reduction in mortality and morbidity among patients suffering from PAD.

描述(申请人提供)：超过800万美国人患有外周动脉疾病(PAD)，通常是由于肢体外周动脉的动脉粥样硬化性闭塞症，导致缺血性溃疡或坏疽等症状。通过再生内皮细胞来改善肢体血流的生物学方法在治疗PAD方面似乎很有前途。内皮细胞再生的一个潜在细胞来源是胚胎干细胞(ESCs)，它是一种可以分化为内皮细胞(ECs)的多潜能干细胞。然而，由于低收益率(通常不到5%)，直接分化为内皮细胞的能力仍然具有挑战性。已知ESC的分化受条件培养液中的生长因子和它们生长的基质的影响。关于确定ESC分化的最佳基质组成，我建议利用细胞外基质(ECM)微阵列。这是一种高通量的方法，用于筛选数百种特定成分的ECM和ECM混合物，用于基质介导的ESC向EC谱系的分化。因此，本项目的目标是优化有利于ESC分化为ECs的基质条件，然后在体内外鉴定ESC来源的ECs的表型和血管生成特性。首先，我将使用高通量的ECM微阵列方法在体外鉴定ESC来源的ECs的分化。微阵列将由含有单一或多种基质蛋白混合物的微型斑点组成。为了便于纯化和非侵入性跟踪，我将使用慢病毒载体转导的ESCs，慢病毒载体由EC特异性启动子VE-cadherin组成，包含编码萤火虫荧光素酶的基因，用于生物发光成像和红色荧光蛋白用于免疫荧光跟踪。接下来，我将在ECM阵列上对ESCs进行分化，并选择用于大规模分化实验的优化EC分化的基质成分。然后，我将通过荧光激活的细胞分选来纯化内皮细胞，并对细胞的表型形态和功能进行表征。最后，利用PAD(后肢缺血)的体内模型，通过生物发光跟踪和激光多普勒血流测量，确定ESC来源的ECs是否能够在缺血肢体中存活，整合到血管系统中，并改善功能。综上所述，该项目将利用创新的策略，促进胚胎干细胞定向分化为内皮细胞，用于PAD患者的血管再生。这项研究对公共健康的潜在影响是降低PAD患者的死亡率和发病率。

项目成果

A matrix micropatterning platform for cell localization and stem cell fate determination.

• DOI: 10.1016/j.actbio.2010.06.033
• 发表时间: 2010-12
• 期刊: ACTA BIOMATERIALIA
• 影响因子: 9.7
• 作者: Huang, Ngan F.;Patlolla, Bhagat;Abilez, Oscar;Sharma, Himanshu;Rajadas, Jaykumar;Beygui, Ramin E.;Zarins, Christopher K.;Cooke, John P.
• 通讯作者: Cooke, John P.