Human tissue-engineered blood vessels using induced pluripotent stem cells

描述（由申请人提供）：由血管堵塞引起的血管疾病是发达国家最大的死亡原因。自体或合成血管移植物用于治疗这种疾病。然而，一些患者要么缺乏合适的自体组织，要么由于靶血管尺寸小而无法接受合成移植物。利用从原代组织中分离的血管平滑肌细胞（SMC）培养的组织工程血管（TEBV）具有很大的潜力，可作为受影响的手术替代工具。 这些患者的血管。然而，使用SMC的用于临床应用的自体TEBV的开发受到患者血管SMC的有限可及性、细胞培养物中SMC分化的快速丧失和原代SMC扩增能力有限的阻碍。因此，建立一种基于人细胞的模型是非常有意义的，该模型为TEBV的建立提供了丰富且可再生的功能性SMC来源。人细胞的可再生来源可以由人诱导多能干细胞（hiPSC）产生，其类似于人胚胎干细胞（hESC）并且可以通过强制基因表达源自人自身的体细胞。hiPSC和hESC都可以自我更新并分化成人体中几乎所有的细胞类型，包括功能性血管SMC，为产生TEBV治疗血管疾病提供了理想的细胞来源。我们最近建立了hiPSC系，并从hiPSC（hiPSC-SMC）和hESC（hESC-SMC）衍生出无限量的高度同质的功能性血管SMC。由于这些iPSC中重编程转基因的潜在再活化可能最终影响其作为治疗的安全性和在疾病建模中的效用，我们将通过使用Cre重组酶产生和验证无转基因的hiPSC系，然后从这些无转基因的hiPSC系衍生和表征hiPSC-SMC。TEBV通常缺乏弹性蛋白（ELN），这对血管的机械性能至关重要，提供了对动脉瘤和扩张的反冲和抵抗力。由于我们已经表明抑制微小RNA-29 a（miR-29 a）显著增加ELN的表达、ELN纤维的交联和源自原代SMC的TEBV的扩张性，我们将在miR-29 a抑制剂存在下使用hiPSC-SMC和hESC-SMC产生TEBV，然后确定TEBV的缝合线保持强度、破裂压力、胶原蛋白含量、ELN含量和机械性质。为了研究SMC衍生的TEBV在体内的功能，我们将TEBV作为主动脉间置移植物植入裸大鼠中。我们选择大鼠模型，因为其他首选的大型动物（狗或猪）可能会拒绝人体组织，即使有免疫抑制，由于显着的异种反应。尽管非人灵长类动物模型不适合用于这种新的hiPSC技术的首次体内研究，但如果啮齿动物模型成功，则其可以在未来与免疫抑制一起用作后续模型。我们将检验以下假设：源自hiPSC-SMC和hESC-SMC的TEBV具有适合于体外植入的性质，然后在体内大鼠主动脉模型中保持机械稳定。

DESCRIPTION (provided by applicant): Vascular disease, caused by the blockage of blood vessels, is the largest cause of mortality in the developed world. Autologous or synthetic vascular grafts are used in treating this disease. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts due to the small size of target vessels. Tissue-engineered blood vessels (TEBVs) grown using vascular smooth muscle cells (SMCs) isolated from primary tissue hold great potential as tools for surgical replacement of the affected vessels in these patients. However, the development of autologous TEBVs for clinical application using SMCs has been hampered by limited accessibility to patient vascular SMCs, rapid loss of SMC differentiation in cell culture and limited ability of primary SMCs to expand. Thus, it is of great interest to establish a human cell-based model that provides an abundant and renewable source of functional SMCs for the establishment of TEBVs. A renewable source of human cells can be generated by human induced pluripotent stem cells (hiPSCs), which resemble human embryonic stem cells (hESCs) and can be derived from a person's own somatic cells by forced gene expression. Both hiPSCs and hESCs can self-renew and differentiate into virtually every cell type in the human body including functional vascular SMCs, providing ideal cell sources for generating TEBVs to treat vascular diseases. We recently established hiPSC lines and derived unlimited amounts of highly homogeneous functional vascular SMCs from hiPSCs (hiPSC-SMCs) and hESCs (hESC-SMCs). As the potential reactivation of reprogramming transgenes in these iPSCs could ultimately affect their safety as therapy and utility in disease modeling, we will generate and validate transgene-free hiPSC lines by using Cre recombinase and then derive and characterize hiPSC-SMCs from these transgene-free hiPSC lines. TEBVs typically lack elastin (ELN), which is essential to mechanical properties of blood vessels, providing recoil and resistance to aneurysm and dilation. Since we have shown that inhibition of microRNA-29a (miR-29a) markedly increases the expression of ELN, crosslinking of ELN fiber and distensibility of TEBVs derived from primary SMCs, we will generate TEBVs using hiPSC-SMCs and hESC-SMCs in the presence of a miR-29a inhibitor and then determine the suture retention strength, burst pressure, collagen content, ELN content, and mechanical properties of TEBVs. To investigate the function of SMC-derived TEBVs in vivo we will implant TEBVs as aortic interpositional grafts in nude rats. We choose the rat model since other preferred large animals (dog or pig) might reject the human tissue even with immunosuppression due to a significant xenogenic response. Although a non-human primate model is not appropriate for a first in vivo study of such new hiPSC technology, it could be used in the future with immunosuppression as a follow-up model if the rodent model succeeds. We will test the hypothesis that TEBVs derived from hiPSC-SMCs and hESC-SMCs possess suitable properties for implantation in vitro and then remain mechanically stable in a rat aortic model in vivo.