Proof of pluripotency of rat iPSCs missing.

We found the article (1) by Drs. G. Li, Y. Chunxu andL. Guisheng, published in February 2013 issue of CellProliferation, to raise several questions and we wouldwelcome a number of points of clariﬁcation from theauthors. In the article, the authors describe reprogram-ming Fawn Hood Hypertensive (FHH) rat embryonic ﬁ-broblasts (REFs), using a piggyBac transposon andtransposase, to introduce genes Oct4, Sox2, Klf4, Myc,(OSKM) or Oct4, Sox2, Klf4, Myc and Lin28 (OSKML),which are linked by sequences encoding 2A peptidesand driven by the CAG promoter, to produce inducedpluripotent stem cells (iPSCs). While the authors claimto have produced iPSCs, their data did not show germ-line competence of the reprogrammed cells. For that rea-son, we think, it is premature to call these rat cellsiPSCs.The authors did not report whether rat or mouseOSKM or OSKML sequences were used in their vector.While they did not report reprogramming efﬁciency,they showed ﬁve small and one large colony in one ﬁeld(Fig. 1c), which was seen on day 10. This would sug-gest high reprogramming efﬁciency and reprogramming,that occurred rapidly. In Fig. 1d, readers are unable tosee alkaline phosphatase (AP) staining, and we ask:what proportion of the colonies stained for AP?In their iPSC characterization, immunocytochemicalstaining of SSEA-1, Oct4, Nanog and Sox2 was shownin Fig. 2; however, readers are unable to verify co-local-ization of these transcription factors in Fig. 2. Stainingin Fig. 2 appears to be from colonies selected to expressthe pluripotency genes. We note that phase contrast andnuclear staining micrographs were not provided. Readersare unable to conﬁrm whether an entire colony stainedfor transcription factors Oct4, Nanog and Sox2 and mosttroubling, the Oct4 staining appears to be cytoplasmicand not nuclear, for many cells.As mentioned above, readers were not told whetherrat or mouse OSKM or OSKML sequences were usedin their piggyBac transposon vector for reprogramming,thus, we were unsure how to interpret the authors’ claimthat endogenous pluripotency markers were expressed(Fig. 2b). How did the authors conﬁrm transfection ofthe piggyBac vector? In Fig. 2b, the authors reportedRT-PCR analysis of endogenous pluripotency markerKlf4, but it was not shown. From information provided,we are able to verify that Nanog was induced, but weare uncertain whether Oct4 and Sox2 expression was theresult of the exogenous vector or of expression ofendogenous genes.According to the Materials and methods section,the authors here injected FHH iPSCs into blastocystscollected from FHH X SD F1 rats. In the Results sec-tion, however, they indicated using FHBN14 X SDblastocysts. Using a GOOGLE Search, FHBN14 ratswere not found. Did they mean FHH-Chr 14BN/Mcwi,for example, FHH rats containing the Brown Norwaychromosome 14? We were confused by Fig. 4b and4d, which showed FHH rat iPSC ‘chimeras’ with adark coat colour. The FHH rat is white with a ‘lighttan’ coloured hood. Would the authors explain why the‘chimeras’ had dark coat colour? Figure 4e does notmake sense either. If we assume that the authors usedFHH-Chr 14BN X SD blastocysts and injected FHHiPSCs, microsatellite analysis would distinguish chima-eras made of FHH iPSCs in the FHH-Chr 14BNxSDF1 host. On the other hand, if they used FHH x SDblastocysts, they would be unable to use microsatelliteanalysis to identify chimaera. If FHH-Chr14BNxSD F1blastocysts were transferred, however, they could nothave an SD littermate (as indicated in the Fig. 4).Finally, according to the Rat Genome Database (http://www.rgd.mcw.edu/rgdweb/report/marker/main.html?id=36520), microsatellite D14Rat7 differentiatesbetween BN rats (174), SD (166) and FHH (142). Werequest clariﬁcation from the authors on these points,please.From our experience with embryo transfer (2), webelieve, it is unlikely that blastocysts transferred intothe uterus of 0.5 days post-coitus (dpc) pseudopregnantfemales would implant and be carried to term. Weinvite the authors to comment on their rationale forembryo transfer into 0.5 dpc females. Do the authorsbelieve that blastocyst transfer into 0.5 dpc pseudopreg-nant females is more efﬁcient than transfer into3.5 dpc pseudopregnant females (as we and othershave performed)?In summary, we cannot agree with the authors’ con-clusion that pluripotency of their iPSCs has been dem-