Abstract
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.
Highlights
Pluripotent stem cells, such as embryonic stem cells (ESCs), are defined by their ability to selfrenew and differentiate into any somatic cell type
Exome sequencing on all 24 induced pluripotent stem cells (iPSCs) clones, as well as the two parental fibroblast pools from which they were derived, was performed to comprehensively define the genetic heterogeneity within iPSCs generated from the same mouse at the same time
To more comprehensively address the degree of genetic heterogeneity that can result from iPSC generation, we characterized the exomic mutations in 24 iPSCs generated from the same C57BL/6 male mouse, using two independent preparations of skin fibroblasts
Summary
Pluripotent stem cells, such as embryonic stem cells (ESCs), are defined by their ability to selfrenew and differentiate into any somatic cell type. In 2006, Yamanaka and colleagues successfully reprogrammed mouse somatic cells into pluripotent stem cells, referred to as induced pluripotent stem cells (iPSCs), by introducing a combination of four transcription factors: Oct3/4, Sox, c-Myc, and Klf4[1]. Using whole genome sequencing to characterize a small number of mouse iPSCs derived from the same animal, Young et al found a number of shared variants between individual iPSC clones (from one of three experiments) that could be detected in
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