Double-Reporter Induced Pluripotent Stem Cells for Vascular Cell Sheet Engineering

用于血管细胞片工程的双报告基因诱导多能干细胞

• 批准号: 9175939
• 负责人: George Kwong
• 金额: $ 3.15万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-02 至 2017-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9175939
• 关键词: Adult; Affect; Allogenic; Antigens; Arteries; Atomic Force Microscopy; Autologous; Biochemical; Blood Vessels; Bypass; Calcium; Cardiac Myocytes; Cell Line; Cells; Characteristics; Clinical; Coculture Techniques; Collagen; Coronary heart disease; Development; DsRed; Endothelial Cells; Engineering; Environment; Epithelial Cells; Evaluation; Extracellular Matrix; Fibroblasts; Fluorescence; Gene Expression; Gold; Green Fluorescent Proteins; Harvest; Heterogeneity; Histocompatibility Testing; Implant; In Vitro; Intercellular Junctions; Lentivirus Vector; Light; Location; Lung; Mechanics; Methodology; Methods; Myosin Heavy Chains; Operative Surgical Procedures; Organ; PECAM1 gene; Patients; Pattern; Phenotype; Polymers; Population; Primitive Streaks; Property; Proteins; Reporter; Role; Site; Smooth Muscle; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Somatic Cell; Sorting - Cell Movement; Source; Staging; Stem cells; Structure; Surface; System; Tail; Temperature; Testing; Thrombosis; Tissue Engineering; Tissues; Traction; Transforming Growth Factor beta; Transgenic Mice; Tubular formation; Vascular Endothelial Growth Factors; Vascular Graft; Vascular Smooth Muscle; Venous; adult stem cell; cadherin 5; cell type; immunogenicity; improved; in vivo; induced pluripotent stem cell; platelet-derived growth factor BB; pluripotency; precursor cell; pressure; promoter; public health relevance; red fluorescent protein; relating to nervous system; self renewing cell; transcription factor; uptake

 DESCRIPTION (provided by applicant): Autologous venous or arterial grafts remain the gold standard therapy for patients with coronary heart disease; however, suitable replacement vessels require multiple surgical sites for harvesting and are often already compromised. Synthetic grafts and tissue-engineered blood vessels (TEBVs) have overcome some of these limitations and have also sought to avoid issues with thrombosis or occlusion. Previous studies have used primary cells and adult stem cells to mimic the native blood vessel structure in TEBVs; however, immunogenicity is still a problem. The recent breakthrough of reprogramming adult somatic cells to induced pluripotent stem cells (iPSCs) provides an alternative cell source, where non-immunogenic cells of multiple lineages can be derived from patient-specific iPSCs. We propose to construct an entirely cellular TEBV using cell sheets of differentiated iPSCs for replacing diseased or damaged arteries. We will develop an iPSC line with a dual reporter for pure populations of differentiated endothelial cells (ECs) and vascular smooth muscle cells (vSMCs). To further our understanding of developmental stages, iPSCs will be differentiated from posterior primitive streak to Flk-1-positive vascular cell precursors to either ECs (using VEGF) or vSMCs (using platelet derived growth factor BB (PDGF-BB) and transforming growth factor beta (TGF-ß)). ECs can be sorted using CD31 (PECAM) and CD144 (VE-Cadherin), and we can use our double reporter system to isolate vSMCs, which are characterized by expression of red fluorescent protein (DsRed) for mural cell marker neural/glial antigen 2 (NG2) and green fluorescent protein (GFP) for smooth muscle actin (SMA). Using a thermoresponsive system, differentiated iPSCs can be formed into cell sheets and detached enzymatically without affecting cell sheet integrity. To study how SMC tissue heterogeneity arises, cell sheets of SMCs will be co-cultured with cell sheets of different tissue types (e.g., airway, cardiomyocytes, or ECs) and evaluated for gene expression (SMA, myosin heavy chain, and SM22a) and functional properties (contractility and calcium uptake). Cell sheets will also be evaluated for mechanical strength and biochemical composition for comparison to native vessel mechanics. With this proposed study, we will lay the groundwork for in vivo evaluation of the functional characteristics of our fabricated TEBV implant to replace damaged blood vessels.

 描述（通过应用程序证明）：自体静脉或动脉移植是具有冠状动脉欠款的金标准治疗，Suithirele Murgical Encore grounder theught of Sue of Aready均经常遭受质量。 Thombosis或闭塞。对于分化LLS（EC）和血管平滑肌细胞（VSMC）的双重报道，以进一步了解我们对发育阶段的理解，ISC将与后原始条纹与FLK-1阳性血管细胞的不同使用VEGF）或VSMCS B（PDGF-BB）和转化生长因子β（TGF-ß）可以使用CD31（PECAM）和CD144（VE-Cadherin）进行排序，我们可以使用我们的双系统来隔离我们的双系统。其特征是用于壁式细胞标记神经/神经胶质抗原2（NG2）和绿色荧光蛋白（GFP）的红色蛋白质（DSRED） SMC组织异质性发生，SMC的细胞表将与不同类型的表（例如气道，心肌细胞或ECS）共同培养，并评估用于基因表达的基因和SM22A中的基因表达）和功能性能（收缩性和钙）机械强度和生化的生化。