University of Cambridge, Cambridge, UK.
Stem Cells Transl Med. 2012 Dec;1(12):855-65. doi: 10.5966/sctm.2012-0093. Epub 2012 Nov 29.
Induced pluripotent stem cells (iPSCs) have the potential to generate patient-specific tissues for disease modeling and regenerative medicine applications. However, before iPSC technology can progress to the translational phase, several obstacles must be overcome. These include uncertainty regarding the ideal somatic cell type for reprogramming, the low kinetics and efficiency of reprogramming, and karyotype discrepancies between iPSCs and their somatic precursors. Here we describe the use of late-outgrowth endothelial progenitor cells (L-EPCs), which possess several favorable characteristics, as a cellular substrate for the generation of iPSCs. We have developed a protocol that allows the reliable isolation of L-EPCs from peripheral blood mononuclear cell preparations, including frozen samples. As a proof-of-principle for clinical applications we generated EPC-iPSCs from both healthy individuals and patients with heritable and idiopathic forms of pulmonary arterial hypertension. L-EPCs grew clonally; were highly proliferative, passageable, and bankable; and displayed higher reprogramming kinetics and efficiencies compared with dermal fibroblasts. Unlike fibroblasts, the high efficiency of L-EPC reprogramming allowed for the reliable generation of iPSCs in a 96-well format, which is compatible with high-throughput platforms. Array comparative genome hybridization analysis of L-EPCs versus donor-matched circulating monocytes demonstrated that L-EPCs have normal karyotypes compared with their subject's reference genome. In addition, >80% of EPC-iPSC lines tested did not acquire any copy number variations during reprogramming compared with their parent L-EPC line. This work identifies L-EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology.
诱导多能干细胞（iPSC）有可能为疾病建模和再生医学应用生成患者特异性组织。然而，在 iPSC 技术能够进入转化阶段之前，必须克服几个障碍。这些障碍包括对理想的体细胞重编程类型的不确定性、重编程的低动力学和效率，以及 iPSC 与其体细胞前体之间的核型差异。在这里，我们描述了使用晚期外生性内皮祖细胞（L-EPC）作为生成 iPSC 的细胞基质的方法。我们已经开发了一种从外周血单个核细胞制剂（包括冷冻样本）中可靠分离 L-EPC 的方案。作为临床应用的原理验证，我们从健康个体和遗传性和特发性肺动脉高压患者中生成了 EPC-iPSC。L-EPC 呈克隆性生长；具有高增殖性、可传代性和可储存性；与成纤维细胞相比，具有更高的重编程动力学和效率。与成纤维细胞不同，L-EPC 高效的重编程允许在 96 孔格式中可靠地生成 iPSC，这与高通量平台兼容。L-EPC 与供体匹配的循环单核细胞的比较基因组杂交分析表明，与参考基因组相比，L-EPC 的核型正常。此外，与亲本 L-EPC 系相比，在重编程过程中超过 80%的 EPC-iPSC 系没有获得任何拷贝数变异。这项工作确定 L-EPC 是 iPSC 生成的实用且高效的细胞基质，有可能解决目前限制该技术转化的许多因素。
