Positive Correlation Between the Efficiency of Induced Pluripotent Stem Cells and the Development Rate of Nuclear Transfer Embryos When the Same Porcine Embryonic Fibroblast Lines Are Used As Donor Cells

Abstract

Induced pluripotent stem cells (iPSCs) and nuclear transfer (NT) are two of the primary routes to reprogram differentiated cells back to the pluripotent state. However, it is still unknown whether there is any correlation between the reprogramming efficiency of iPSCs and NT if the same donor cells are employed. In this study, six porcine embryonic fibroblast (PEF) lines from Landrace (L1, L6, L9) or Congjiang local pigs (C4, C5, C6) were used for iPSC induction and NT. Furthermore, the resultant iPSCs from four PEF lines (L1, L6, C4, and C5) were used for NT (iPSC-NT), and the expression of exogenous genes was detected in iPSC-NT embryos by real-time PCR. The results showed that the efficiency of iPSC lines established from different PEF lines were significantly different. When the same PEF lines were used as donor cells for NT, the blastocysts rates were also different among different PEF lines and positively related with iPSCs induction efficiency. When the iPSCs were used as donor cells for NT, compared with the source PEFs, the blastocysts rates were significantly decreased. Real-time PCR results indicated that exogenous genes (Oct4, c-Myc) continued to be expressed in iPSC-NT embryos. In summary, our results demonstrate that there was a positive correlation between iPSCs and NT reprogramming efficiency, although the mechanism of these two routes is different. This may provide a new method to select the appropriate donor cells for inducing iPSCs.

Ovine Induced Pluripotent Stem Cells Are Resistant to Reprogramming after Nuclear Transfer

“…This finding is consistent with a recent publication reporting similar developmental competence of pig iPSCs and fibroblasts (Xie et al, 2014). While this manuscript was in preparation, a report showed that iPSCs synchronized in G 2 /M are better suited for NT compared to G 1 and unsynchronized cells (Yuan et al, 2014).…”

“…4 were cultured with FBS (as were the cloned embryos). As with endogenous SOX2, we observed no silencing of exogenous pluripotency genes during preimplantation development of sheep iPS-NT embryos, which is consistent with the recent publication in pigs (Xie et al, 2014).…”

“…Persistent expression of transgenes and increased number of aneuploidies in donor cells may be the reason for compromised reprogramming and developmental potential NT embryos, although the underlying reason for these has yet to be determined. Nonetheless, from our results in sheep and from those reported by other groups in pigs (Fan et al, 2013;Xie et al, 2014), it is plausible to propose that iPSCs generated by non-integrating methods (free of transgenes) would be better candidates for the production of transgenic animals by NT.…”

Exploring differentially expressed genes related to metabolism by RNA-Seq in porcine embryonic fibroblast after insulin treatment

“…The PEF lines were derived from the Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, and the PEF culture method has been published [ 13 ]. In short, prior to add human insulin (0.5 ug), PEF lines were thawed and cultured, and the cells were collected at 48 h and 72 h after the addition of insulin.…”

Pluripotent stem cells and livestock genetic engineering

“…While in mice production of cloned animals from iPSCs has similar efficiency to that of ESCs (Kou et al 2010; Zhou et al 2010), attempts to clone pigs and sheep from iPSCs have yielded very low efficiencies (Fan et al 2013b; German et al 2015; Xie et al 2014). A large effort to clone from pig iPSCs only proved successful when the iPSC were induced to differentiate before the NT procedure (Fan et al 2013b).…”