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

 描述(申请人提供)：自体静脉或动脉移植仍然是冠心病患者的黄金标准治疗方法；然而，合适的替代血管需要多个手术部位才能获取，而且往往已经受到损害。合成移植物和组织工程血管(TEBV)已经克服了其中的一些限制，并试图避免血栓形成或闭塞的问题。以前的研究已经使用原代细胞和成体干细胞来模拟TEBV的天然血管结构；然而，免疫原性仍然是一个问题。最近对成体细胞重新编程为诱导多能干细胞(IPSCs)的突破提供了另一种细胞来源，其中多个谱系的非免疫原性细胞可以从患者特定的IPSCs中获得。我们建议使用分化的IPSCs细胞片构建一个全细胞的TEBV，用于替换病变或受损的动脉。我们将开发一种具有双重报告基因的IPSC系，用于分化的内皮细胞(ECs)和血管平滑肌细胞(VSMCs)的纯群体。为了加深我们对发育阶段的理解，IPSCs将从后部原始条纹到Flk-1阳性的血管细胞前体分化为内皮细胞(使用血管内皮生长因子)或血管平滑肌细胞(使用血小板衍生生长因子BB和转化生长因子β)。内皮细胞可用CD31(PECAM)和CD144(VE-钙粘附素)分选，我们可以用我们的双报告系统分离vSMCs，其特征是表达壁细胞标志神经/神经胶质抗原2(NG2)的红色荧光蛋白(DsRed)和表达平滑肌肌动蛋白(SMA)的绿色荧光蛋白(GFP)。使用热响应系统，分化的IPSCs可以形成细胞膜，并在不影响细胞膜完整性的情况下以酶法分离。为了研究SMC组织异质性是如何产生的，SMC的细胞片将与不同组织类型的细胞片(例如，呼吸道、心肌细胞或内皮细胞)共同培养，并评估基因表达(SMA、肌球蛋白重链和SM22a)和功能特性(收缩和钙摄取)。还将评估细胞片的机械强度和生化成分，以与本地血管力学进行比较。通过这项拟议的研究，我们将为体内评估我们制作的TEBV植入物以取代受损血管的功能特征奠定基础。

 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.