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造成的有限耐用性的一个策略是合成膜表面的功能化。化学修饰或包覆生物物质如肝素已被用于减少血栓细胞粘附。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以创造一个天然的非血栓形成表面，这可以允许长期使用膜氧合器而不会形成血栓。虽然针对气体交换膜内皮化的初步研究显示出令人鼓舞的结果，但适合临床规模和应用的内皮细胞（EC）来源尚未确定。对于膜表面约为1.3 - 4平方米的典型膜氧合器的播种，需要具有高膨胀电位的细胞源，可以提供具有天然EC层细胞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氧合器的发展确实忽略了这些需求，以及最近的报道表明，在培养的原发性内皮细胞中经常出现染色体畸变。由于成人原代ECs（例如来自隐静脉）缺乏足够的可扩展性，大多数实验研究使用从人脐带血或脐静脉分离的ECs。基于我们之前的研究结果，我们假设患者特异性ipsc衍生的内皮细胞是膜氧合器内皮化的最合适细胞来源。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内皮细胞的可扩展性、遗传完整性和功能（来自脐带血和成人外周血的晚期内皮细胞、来自脐静脉和成人隐静脉的内皮细胞、来自脂肪组织的微血管内皮细胞以及从人类iPSCs分化的内皮细胞）。特别是，我们将研究细胞功能和分泌特性在延长培养扩增后的衰老相关变化，这将需要提供每个设备所需的大约10到20亿个细胞的细胞数量。因此，该项目将对生物功能化膜氧合器和其他基于ec的细胞疗法的临床翻译做出重要贡献。

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.