Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13NEGSSEA4POSTra-1-60POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.当前通过病毒感染从人真皮成纤维细胞（human dermal fibroblasts）诱导获得诱导多能干细胞（induced pluripotent stem cell, iPSC）株系的方法，均依赖于成本高昂且周期冗长的实验方案。导致该建系流程耗时较长的核心瓶颈之一，是研究人员难以在感染后的早期时间点，从混杂有转化细胞、部分重编程细胞与成纤维细胞的混合细胞群中，精准鉴定出完全重编程的特异性候选克隆。若在建系初期未能筛选出高质量的细胞集落，后续获得的细胞株系将需要更频繁的维护，且实验结果的可靠性将大幅降低。本研究报道了一种优化的iPSC株系构建方法：借助荧光激活细胞分选术（fluorescence activated cell sorting, FACS），在感染后第7至10天，分选表达细胞表面标志物特征CD13NEGSSEA4POSTra-1-60POS的单个细胞。该技术可前瞻性地分离得到完全重编程的iPSC，同时去除亲本细胞与“污染性”的部分重编程成纤维细胞，从而在无需使用特定小分子组合的前提下，大幅缩短iPSC株系构建所需的时间并减少试剂用量。经FACS分选获得的iPSC株系可表达多能性相关的经典标志物，并具备体外与体内的自发分化潜能。为验证FACS技术用于高通量iPSC构建的适用性，我们分别采用整合型（逆转录病毒）与非整合型（仙台病毒（Sendai virus））重编程载体，成功构建了228株独立的iPSC株系，并对其中部分株系开展了全面的表征分析。本研究中使用的iPSC株系，均源自76份来自不同组织来源的独特样本，包括从健康受试者或疾病患者的活检组织中获取的新鲜或冻存成纤维细胞。