Genetic Heterogeneity of Induced Pluripotent Stem Cells: Results from 24 Clones Derived from a Single C57BL/6 Mouse

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs “captures” the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically “fit” for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0–45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40–60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.诱导多能干细胞（induced pluripotent stem cells, iPSCs）作为疾病建模、药物筛选及其他相关应用的工具，具备极高的应用潜力。由于iPSC的生成过程会“捕获”被重编程的单个细胞的遗传背景，即便源自同一供体的iPSC克隆也会呈现出遗传异质性。为评估遗传异质性的程度，并探究是否部分细胞在重编程过程中具备更优的遗传“适应性”，我们对24株小鼠iPSC克隆开展了外显子组测序（exome sequencing），这些克隆的原材料为从同一只8周龄C57BL/6J雄性小鼠的两个不同皮肤部位分离得到的皮肤成纤维细胞（skin fibroblasts）。尽管两个亲本成纤维细胞群体的编码区域未检测到任何差异，但每株克隆均拥有独特的遗传特征，各克隆间的异质性跨度较大：针对这24株克隆，我们共验证得到383个iPSC相关变异位点，平均每株克隆包含16.0个变异位点，变异范围为0~45。由于这些变异在每株克隆的绝大多数细胞中均存在（杂合变异的变异等位基因频率介于40%~60%之间），它们大概率预先存在于被重编程的单个细胞中，而非重编程过程或细胞传代阶段新获得的变异。随后我们通过体外诱导生成造血祖细胞（hematopoietic progenitors）并计数集落形成单位（colony forming units, CFUs），分析了该遗传异质性是否会对造血发育产生功能性影响。尽管24株克隆的造血潜能存在一定差异，但仅1株克隆无法分化为造血细胞；不过该克隆可形成畸胎瘤（teratoma），证实了其多能性本质。此外，未发现突变谱与各iPSC克隆的造血潜能之间存在特异性关联。上述数据清晰揭示了iPSC生成过程所捕获的、存在于单个成纤维细胞内的遗传异质性，并提示绝大多数此类变异为随机发生且功能上良性的。