Ectopic expression of reprogramming factors has been widely adopted to reprogram somatic nucleus into a pluripotent state (induced pluripotent stem cells [iPSCs]). However, genetic aberrations such as somatic gene mutation in the resulting iPSCs have raised concerns regarding their clinical utility. To test whether the increased somatic mutations are primarily the by-products of current reprogramming methods, we reprogrammed embryonic fibroblasts of inbred C57BL/6 mice into either iPSCs (8 lines, 4 previously published) or embryonic stem cells (ESCs) with somatic cell nuclear transfer (SCNT ESCs; 11 lines). Exome sequencing of these lines indicates a significantly lower mutation load in SCNT ESCs than iPSCs of syngeneic background. In addition, one SCNT-ESC line has no detectable exome mutation, and two pairs of SCNT-ESC lines only have shared preexisting mutations. In contrast, every iPSC line carries unique mutations. Our study highlights the need for improving reprogramming methods in more physiologically relevant conditions.

In this study, we characterized the protein-coding region of 8 iPSC lines and 11 SCNT-ESC lines derived from a syngeneic inbred mouse background at single-nucleotide resolution. We chose to focus on exome not only due to cost considerations but also because exome mutations are more interpretable, and the iPSC mutation load in exome has been well characterized (). We observed significantly lower somatic-coding mutation load in SCNT ESCs than iPSCs. These findings suggest that current reprogramming methods to generate iPSCs could be improved in more physiologically relevant conditions to optimize nuclear reprogramming for clinical application.

SCNT has been widely used to reprogram the somatic nucleus into a pluripotent state by the injection of a donor nucleus into an enucleated oocyte. The technique mimics the process of early embryonic development except that the blastocyst formed contains identical genomic DNA as the donor ( Figure 1 ). Because SCNT provides a physiologically relevant condition for nuclear reprogramming and allows development, it has been widely adopted to produce viable cloned mammals such as sheep (), mice (), and rabbit () from primary culture. There is also an ongoing effort to derive human ESC-like lines using SCNT (SCNT ESCs) for patient-specific therapy (). However, the genetic integrity of these SCNT ESCs has yet been reported.

Reprogramming of somatic nuclei into a pluripotent state can be achieved through either somatic cell nuclear transfer (SCNT) () or ectopic expression of reprogramming factors in somatic cells to generate induced pluripotent stem cells (iPSCs) (). The latter approach has become widely adopted because it is ethically more acceptable and technically more feasible to many organisms such as humans. The iPSCs are functionally indistinguishable from embryonic stem cells (ESCs). However, recent studies have revealed genetic and epigenetic aberrations in the resulting iPSCs (). For example, it has been shown that iPSCs always possess somatic-coding mutations (), leading to the concern for the safety of such cells in clinical application.

In contrast to the findings that every iPSC line examined in this and previous studies harbored protein-coding mutations, we did not detect any protein-coding mutation in one SCNT-ESC line ( Table 1 , SCNT2). Moreover, there were two pairs of SCNT-ESC lines (SCNT3 and SCNT4 and SCNT6 and SCNT10) that only had shared somatic mutations ( Table 2 ), suggesting that these mutations were not introduced during reprogramming but, instead, were present in the fibroblast progenitors. In SCNT-ESC lines, the blastocyst formed contains identical genomic information as the donor ( Figure 1 ). Given the nature of this technique, any shared mutations in SCNT ESCs derived from the same donor cells were possibly inherited from a rare parental fibroblast carrying these mutations. None of these mutations located in any of the known mutation hot spots, so the possibility of seeing two mutations occurring at the same position due to “mutation hot spots” was too low even for one line. After removing these potentially preexisting shared mutations, four SCNT-ESC lines carried no detectable coding mutations introduced during reprogramming. In contrast, all mutations identified in the iPSC lines were unique. We observed great variability in somatic-coding mutational load across SCNT-ESC and iPSC lines that contributed to chimeric mice. Therefore, these detectable mutations appear to have no apparent functional consequence in development. Furthermore, none of the mutated genes clusters in a specific functional pathway.

Previous studies have reported that human iPSCs derived from diverse somatic origins and reprogramming methods all carried between 2 and 14 point mutations in protein-coding regions (). In this study, we sought to determine whether acquisition of protein-coding mutations must occur to allow successful nuclear reprogramming. To avoid the influence of distinct genetic backgrounds on reprogramming, we derived 11 mouse SCNT-ESC lines and 4 mouse iPSC lines from the syngeneic mouse embryonic fibroblasts (MEFs) of the inbred C57BL/6 (B6) mice ( Figure 1 ). The B6 iPSCs were reprogrammed with the integration-free approaches by Abe’s and our group (), and the SCNT ESCs were generated as we previously described (). The pluripotency of these cell lines was extensively characterized by the surface expression of ESC-specific markers, their capability to differentiate into each of the three germ layers, or in most cases, by their capability to contribute to adult chimeric mice and germline transmission ( Figures 2 A–2D; Figures S1 and S2 available online). We next performed exome sequencing and pairwise comparison of the 19 pluripotent stem cell lines and progenitor MEF cells ( Figure 1 ). After mapping sequenced reads, 90% or more of protein-coding regions had sufficiently high sequence coverage (>10×) and consensus quality (>30) to identify somatic-coding mutations in each cell line ( Table 1 ). We identified and validated a total of 78 unique somatic-coding mutations within 8 iPSC lines ( Tables 1 2 , and S1 ), or an average of 9.8 mutations per line, consistent with previously observed mutational load in human iPSC lines (). In contrast, 31 unique mutations were identified and validated in the 11 SCNT-ESC lines, leading to a projection of 2.8 mutations per line in protein-coding regions ( Tables 1 2 , and S1 ). The mutational load of iPSC lines was significantly higher than that of SCNT-ESC lines (p < 0.004, Mann-Whitney U test).

Quality-filtered sequence represents the amount of sequence data generated that passed the Illumina quality filter with a sequencing depth of at least 8 and a consensus quality score of at least 30 (bp). The dbSNP percentage is the percentage of identified variants that are in the Single Nucleotide Polymorphism Database. The shared coding region is the portion of the genome that was sequenced at high depth and quality in both the pluripotent stem cell line and matched fibroblast (as shown in Figure 1 ). The projected number of somatic-coding mutations is calculated by the fraction of consensus coding sequence identified in both pluripotent stem cells and fibroblasts.

(B) Pluripotency of SCNT ESCs in vivo. Left panel shows chimeric mice derived from SCNT-ESC lines. SCNT1 cells were injected into eight-cell embryos of ICR mice, and shown are 8-week-old offspring, in which black coat color is derived from the SCNT-ESC contribution. Right panel shows the male that was crossed with a white ICR female, producing a litter containing nine black offspring, confirming the contribution of SCNT1 to the germline. Asterisks in the left and right panels indicate the same male.

Discussion

The major advantage of using cells from an inbred mouse strain for the comparison of various reprogramming technologies was that the MEFs isolated from these mice were genetically identical, in contrast to the human cell lines that have much more genetic variability. It also enables more comprehensive pluripotent functionality testing such as the contribution to chimeric mice. In addition, genetic difference between inbred mice is usually present as homozygous variants, whereas somatic mutations would always appear as heterozygous. By exome sequencing of SCNT ESCs and iPSCs reprogrammed from syngeneic mouse B6 fibroblasts, we were able to perform a direct comparison of the somatic mutation load between the two nuclear-reprogramming methods.

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et al. Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming. Previous studies reported an average of 6–12 somatic-coding mutations in human iPSC lines when compared against their corresponding somatic cell of origin (). We discovered that, at least in mouse cells, the somatic mutation load in SCNT-ESC lines was significantly lower than that of iPSC lines. Furthermore, one of the SCNT-ESC lines has no detectable coding mutation. Studies have suggested that some but not all identified somatic variants in iPSCs, such as point mutations and copy number variations, were present in their progenitor cells, whereas others were introduced during reprogramming (). Therefore, our findings of two pairs of SCNT-ESC lines that only harbor shared mutations suggest that genetic variants most likely preexisted in the somatic population of origin, without acquiring any additional coding somatic mutation during reprogramming. The differential somatic mutation load in pluripotent stem cells reprogrammed with the two methods could be due to the difference in their derivation time. In this context, the iPSCs are established 2–4 weeks after ectopic expression of transcription factors, whereas SCNT ESCs are established 4 days after oocyte activation. Therefore, it is very likely that formation of iPSCs has to go through additional rounds of cell division and a potentially more stressful condition when compared to SCNT ESCs. Taking into consideration the differential reprogramming time, the iPSCs spent an average of 50–77 days in culture, whereas SCNT ESCs spent an average of 25–32 days in culture. By dividing the average number of mutations observed with the number of days in culture, iPSCs give rise to at least twice the number of mutations per day compared to SCNT ESCs. Although it has been reported that reprogramming associated somatic-coding mutations individually does not provide a selective advantage to facilitate the acquisition of pluripotency during reprogramming, it remains to be determined whether a combination of mutations could have a role in reprogramming (). However, we could not rule out the possibility that, during an extended period of induced pluripotency, somatic mutations might selectively accumulate and/or enrich over time.

Our data suggest that, when compared to induced pluripotency, SCNT might be a safer way to reprogram somatic cells into a pluripotent state with a lower mutation load. Therefore, it is important to optimize the condition of induced pluripotency into a more physiologically relevant context to minimize genetic aberrations and improve the feasibility for clinical application.