Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.
issue of Cell Proliferation, to raise several questions and we would welcome a number of points of clarification from the authors. In the article, the authors describe reprogramming Fawn Hood Hypertensive (FHH) rat embryonic fibroblasts (REFs), using a piggyBac transposon and transposase, to introduce genes Oct4, Sox2, Klf4, Myc, (OSKM) or Oct4, Sox2, Klf4, Myc and Lin28 (OSKML), which are linked by sequences encoding 2A peptides and driven by the CAG promoter, to produce induced pluripotent stem cells (iPSCs). While the authors claim to have produced iPSCs, their data did not show germline competence of the reprogrammed cells. For that reason, we think, it is premature to call these rat cells iPSCs.The authors did not report whether rat or mouse OSKM 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 suggest high reprogramming efficiency and reprogramming, that occurred rapidly. In Fig. 1d, readers are unable to see alkaline phosphatase (AP) staining, and we ask: what proportion of the colonies stained for AP?In their iPSC characterization, immunocytochemical staining of SSEA-1, Oct4, Nanog and Sox2 was shown in Fig. 2; however, readers are unable to verify co-localization of these transcription factors in Fig. 2. Staining in Fig. 2 appears to be from colonies selected to express the pluripotency genes. We note that phase contrast and nuclear staining micrographs were not provided. Readers are unable to confirm whether an entire colony stained for transcription factors Oct4, Nanog and Sox2 and most troubling, the Oct4 staining appears to be cytoplasmic and not nuclear, for many cells.As mentioned above, readers were not told whether rat or mouse OSKM or OSKML sequences were used in their piggyBac transposon vector for reprogramming, thus, we were unsure how to interpret the authors' claim that endogenous pluripotency markers were expressed (Fig. 2b). How did the authors confirm transfection of the piggyBac vector? In Fig. 2b, the authors reported RT-PCR analysis of endogenous pluripotency marker
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