Human tissue-engineered blood vessels using induced pluripotent stem cells

描述(申请人提供)：血管疾病，由血管阻塞引起，是发达国家最大的死亡原因。自体或人造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，要么由于靶血管较小而无法接受合成移植物。从原代组织分离的血管平滑肌细胞(SMCs)培养出来的组织工程血管(TEBV)具有巨大的潜力，可以作为手术替代受影响血管的工具 这些患者体内的血管。然而，由于患者血管SMC的可获得性有限、SMC在细胞培养中分化的迅速丧失以及原代SMC的有限的扩增能力，利用SMC进行临床应用的自体TEBV的发展一直受到阻碍。因此，建立一种基于人类细胞的模型，为TEBV的建立提供丰富和可再生的功能性SMC来源是非常有意义的。人类诱导多能干细胞(HiPSCs)可以产生一种可再生的人类细胞来源，它类似于人类胚胎干细胞(HESCs)，可以通过强制基因表达从人自己的体细胞衍生而来。HiPSCs和hESCs都可以自我更新和分化为人体内几乎所有类型的细胞，包括功能性血管SMCs，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hESCs(hESC-SMC)中获得了无限数量的高度同质的功能性血管SMC。由于这些IPSCs中转基因的重新激活可能最终影响其作为治疗手段的安全性和在疾病建模中的实用性，我们将利用Cre重组酶建立并验证无转基因的HiPSC系，然后从这些无转基因的HiPSC系中获得并鉴定HiPSC-SMC。TEBV通常缺乏弹性蛋白(ELN)，弹性蛋白(ELN)对血管的机械性能至关重要，提供对动脉瘤和扩张的反冲和抵抗。由于我们已经证明抑制microRNA-29a(miR-29a)显著增加了ELN的表达、ELN纤维的交联性和来自原代SMC的TEBV的膨胀性，因此我们将在miR-29a抑制剂的存在下使用HiPSC-SMC和hESC-SMCs产生TEBV，然后测定TEBV的缝合保持强度、破裂压力、胶原含量、ELN含量和机械性能。为了研究SMC来源的TEBV在体内的功能，我们将TEBV作为主动脉间置移植物植入裸鼠体内。我们选择大鼠模型是因为其他首选的大型动物(狗或猪)可能会排斥人类组织，即使由于显著的异种反应而受到免疫抑制。虽然非人类灵长类动物模型不适合于这种新的HiPSC技术的第一次体内研究，但如果啮齿动物模型成功，它可以在未来用于免疫抑制作为后续模型。我们将在体外测试hPSC-SMCs和hESC-SMCs来源的TEBV具有合适的植入特性，然后在体内的大鼠主动脉模型中保持机械稳定性。

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.