129B6F1G1 and GR14 are nuclear transfer-
derived ES(ntES) cell lines (Wakayama et al
2001) previously established in our laboratory
using sertoli cell from a 129B6F1 background
With GFP(Ohta ＆ Wakayama 2005) and
tail tip cell of a mail mous
(129BDF2. Wakayama et al 2005) respectively
as donor for nuclear transfer karyotype
analysis revealed that the ES cell lines
Used had the normal karyotype at the
following percentages (number of metaphases
with normal karyotype in parentheses)
46%(12／26)for E14 35%(9／26) for
129B6F1G1 and54%(14／26)for GR14
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To evaluate whether the iMuSCs could give rise to chimeric mice, a blastocyst injection assay was performed (Fig. 4c). We transferred undifferentiated β-gal+ and GFP-pre-labelled iMuSCs as single cells into BALB/c (Jackson Lab, USA) blastocysts by microinjection following standard procedures[8].
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[8].Nagy, A. Manipulating the mouse embryo: a laboratory manual. 3rd edn, (Cold Spring Harbor Laboratory Press, 2003).
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Expression of embryonic-like phenotypes
Expression of the following genes normally associated with the early stage of embryonic development was seen in cells procured from each tissue. The gene profiles that were expressed are recognized as ESC markers and suggestive of pluripotency. Oct4, Nanog, Zfp296, Cripto, Gdf3, Utf1, Ecat1, Esg1, Sox2, and Fgf5 (Figs. 2 and 3) were expressed in cells procured from each tissue source. In contrast, Rex1, Eras, and Cdx2 were not expressed at the mRNA level (Figs. 2 and 3; Supplementary Table S2), suggesting that although the cells analyzed in the study were very similar to ESCs, they were not identical.
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In quantitative polymerase chain reaction (qPCR) analysis, STAP cells (sorted for strong Oct4-GFP signals) expressed not only pluripotency marker genes but also trophoblast marker genes such as Cdx2 (Fig. 1d and Supplementary Table 1 for primers), unlike ES cells. Therefore, the blastocyst injection result is not easily explained by the idea that STAP cells are composed of a simple mixture of pluripotent cells (Oct4+Cdx2−) and trophoblast-stem-like cells (Oct4−Cdx2+).
