Generation of Genome Integration-free Induced Pluripotent Stem Cells from Fibroblasts of C57BL/6 Mice without c-Myc Transduction

Abstract

Although the induction of genome integration-free induced pluripotent stem cells (iPSCs) has been reported, c-Myc was still required for the efficient generation of these cells. Herein, we report mouse strain-dependent differences in the c-Myc dependence for iPSC generation and the successful generation of genome integration-free iPSCs without c-Myc transduction using C57BL/6 mouse embryonic fibroblasts. We performed 49 independent experiments and obtained a total of 24 iPSC clones, including 18 genome integration-free iPSC clones. These iPSCs were indistinguishable from embryonic stem cells and from iPSCs generated using other methods. Furthermore, the generation of three-factor iPSCs free of virus vectors revealed the contribution of c-Myc to the genomic integration of external genes. C57BL/6 is an inbred mouse strain with substantial advantages for use in genetic and molecular biological studies due to its use in the whole mouse genome sequencing project. Thus, the present series of C57BL/6 iPSCs generated by various procedures will serve as a valuable resource for future genetic studies of iPSC generation.

Crucial Role of C-Myc in the Generation of Induced Pluripotent Stem Cells

“…S1). We also could not distinguish these iPSCs from ES cells [17]. Hence, our initial results were strictly consistent with previously reported findings [3,4].…”

“…We examined 2A-3F-iPSC and 2A-4F-iPSC clones, which were verified previously to be genome integration-free [17]. Few differences were observed between these two types of iPSCs in terms of phenotypic, molecular biological, or cytochemical analyses.…”

“…We observed positive effects of c-Myc transduction upon iPSC generation, most notably on pluripotency, using an integration-free vector from C57BL/6J mice [15,17]. We then conducted similar analyses of iPSCs generated from this inbred strain using another transduction system involving retroviral vectors that has been widely used for this purpose (two of the 4F-iPSC and five of the 3F-iPSC clones).…”

“…We attempted to carefully compare 4F-generated and 3F-generated iPSCs by using newly established cells generated from the inbred mouse strain, C57BL/6J, using a nonviral vector [17]. We anticipated that a common genetic background would allow us to better investigate the role of the introduced c-Myc in iPSCs, that is, with minimal external influences such as the integration of the exogenous genes into the genome through the use of a viral vector, which could potentially induce mutations.…”

“…Having observed that 2A-3F-iPSCs are severely defective in their developmental ability, we initially hypothesized that this aberration was due to an epigenetic control defect and investigated the DNA methylation status of the promoter regions of the stem cell marker genes, Oct3/4, Nanog [17], Dppa2, Rex1, Stella, Utf1, and Esg1 [24]. However, the promoters of these genes were demethylated to a similar extent in 3F-iPSCs and 4F-iPSCs (Supporting Information Fig.…”