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. 2016 Jun 1;6:27059. doi: 10.1038/srep27059

Figure 8. Exogenous reprogramming factors are still expressed but not inserted in genomic DNA of NI13 and NI20 cell lines.

Figure 8

(A) Real-Time PCR were performed on cDNAs produced from NI13 and NI20 cell lines at early (p7, blue bars) and late (respectively p29 and p33, red bars) passages. Expression of exogenous factors hsOCT4 (SeV OCT4), hsSOX2 (SeV SOX2), hs KLF4 (SeV KLF4), hsMYC (SeV MYC) and Sendai virus RNA (SeV) was quantified between early and late passages in culture. Data are means and SD of two independent experiments (t-test, *P < 0.05; **P < 0.01; ***P < 0.001, NS: not significant). (B) PCR analysis was performed on human and pig genomic DNA with primers recognizing specifically the coding sequence of human or pig pluripotency genes. 40 amplification cycles were performed on genomic DNA from human cells or NI13 and NI20 cells at early and late passages. Human sequences were never amplified in the genomic DNA from NI-iPSLCs. (C) Representation of the Sendai virus genome and map of the primers used in the PCR analysis. Primers for detecting reprogramming genes are located on one side in the Sendai genome and on the other side in the coding sequence of reprogramming factors, depending on their insertion sites. Three pairs of primers amplifying Sendai virus genes are also mapped. PCRs were performed on genomic DNA from NI13 and NI20 cell lines at early and late passages for 40 amplification cycles. Insertion of exogenous factors hsOCT4 (SeV OCT4), hsSOX2 (SeV SOX2), hs KLF4 (SeV KLF4), hsMYC (SeV MYC) and Sendai virus genes (SeV, SeV L and SeV HN) was never detected. Oppositely, RT-PCRs using RNAs extracted from NI13 and NI20 cell lines at early and late passages clearly show that the whole Sendai virus RNA is expressed and is maintained in the cytoplasm of pig NI-iPSLCs. hsOCT4 and hsMYC expression is maintained among passages, while hsSOX2 and hsKLF4 are not detectable with this assay. (D) Real-Time PCR were performed on cDNAs produced from NI13 and NI20 cell lines at early (p7, blue bars) and late (respectively p29 and p33, red bars) passages to quantify relative expression of endogenous pluripotency markers ssOCT4, ssSOX2, ssKLF4, ssMYC, ssSALL4, ssNANOG, ssESRRB and ssLIN28. Data are means and SD of two independent experiments (t-test, *P < 0.05; **P < 0.01; ***P < 0.001, NS: not significant).