Patient-specific induced pluripotent stem (iPS) cells for endothelialisation of membrane surfaces of implantable biohybrid lung devices

克服当前肺辅助装置由于凝血和血栓形成而导致的有限耐久性的一种策略是合成膜表面的功能化。已经应用化学修饰或用生物物质如肝素涂覆以减少血小板粘附。虽然这些策略可以在一定程度上改善血液相容性，但气体交换膜的长期功能尚未实现。据推测，血液接触材料的稳定内皮化可能会产生一个天然的非血栓形成的表面，这可能会允许长期使用的膜式氧合器没有血栓形成，而第一次研究旨在气体交换膜的内皮化显示令人鼓舞的结果，内皮细胞（EC）来源适合临床规模和应用尚未确定。为了接种具有约1.3至4平方米的膜表面的典型膜氧合器，具有高扩增潜力的细胞源将是必要的，其可以提供具有天然EC层中细胞的所有典型功能特性的遗传稳定的EC。值得注意的是，最近对内皮化植入式膜式氧合器的开发确实忽视了这些需求，以及最近的报告表明培养的原代EC中频繁发生染色体畸变。由于成人原代内皮细胞缺乏足够的可扩增性（例如来自隐静脉），大多数实验研究使用从人脐带血或脐静脉分离的内皮细胞。基于我们先前的结果，我们假设患者特异性iPSC衍生的内皮细胞代表用于膜氧合器内皮化的最合适的细胞来源。在这里，我们建议进行一项系统的研究，分析来自不同细胞来源的EC的可扩展性，遗传完整性以及功能性（来自脐带血和成人外周血的晚期生长EC，来自脐静脉和成人隐静脉的EC，来自脂肪组织的微血管EC以及从人iPSC分化的内皮细胞）。特别是，我们将研究延长培养扩增后细胞功能和分泌特征与衰老相关的变化，这是每个设备提供约10亿至20亿个细胞所需的细胞数量所需的。因此，该项目将为生物功能化膜式氧合器和其他基于EC的细胞疗法的临床转化做出重要贡献。

One strategy to overcome the limited durability of current lung assist devices due to clotting and thrombus formation is the functionalization of synthetic membrane surfaces. Chemical modification or coating with biological substances such as heparin have been applied to reduce thrombocyte adhesion. Although such strategies can improve the hemocompatibility to a certain extent, long-term functionality of the gas exchange membranes has not been achieved yet. It has been hypothesized that stable endothelialization of blood contacting materials may create a natural non-thrombogenic surface, which could allow for long-term use of membrane oxygenators without thrombus formation.While first studies aiming at the endothelialization of gas exchange membranes show encouraging results, an endothelial cell (EC) source suitable for clinical scale up and application is not yet identified. For seeding of a typical membrane oxygenator with a membrane surface of about 1.3 to 4 sqm, a cell source with high expansion potential will be necessary, that can provide genetically stable ECs with all typical functional properties of cells in the natural EC layer. It is noteworthy that recent developments towards an endothelialized implantable membrane oxygenator did neglect these needs as well as recent reports that demonstrated frequent chromosomal aberrations in cultured primary ECs. Due to the lack of sufficient expandability of adult primary ECs (e.g. from saphena veins), most experimental studies used ECs isolated from human cord blood or umbilical veins.Based on our previous results we hypothesize that patient-specific iPSC-derived ECs represent the most suitable cell source for endothelialization of membrane oxygenators. Here we propose to conduct a systematic study that analyses expandability, genetic integrity as well as functionality of ECs from different cell sources (late outgrowth ECs from cord and adult peripheral blood, ECs from umbilical veins and adult saphena veins, microvascular ECs from adipose tissue as well as endothelial cells differentiated from human iPSCs). In particular, we will investigate senescence-related changes in cell function and secretory characteristics after extended culture expansion that would be required to provide the necessary cell numbers of about 1 to 2 billion cells per device. This project will thus critically contribute to clinical translation of biofunctionalized membrane oxygenators and other EC-based cellular therapies.