A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

Abstract

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.

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

“…The relevance of elevated, somewhat varied expression of several genes encoding transcription factors that may direct trophoblast formation (38-40) (e.g., GATA3, TFAP2A, TFAP2C, CDX2, EOMES) is unclear but might reflect the fact that such cells are hovering on the point of differentiating along that lineage (i.e., transcriptionally primed) as soon as the pluripotent gene networks are down-regulated. Although the observation that CDX2 was coexpressed with POU5F1 in the undifferentiated H1 BP cells might be considered surprising, CDX2 expression has been consistently observed in rat ESCs (41)(42)(43) and associated with POU5F1 expression in human (44) and bovine (45) embryonic trophectoderm. More unexpected was the coexpression of CDX2 with NANOG, because the two transcription factors have mutually antagonistic effects in mouse ESCs and preimplantation embryos (46).…”

Inhibition of Protein Kinase C Signaling Maintains Rat Embryonic Stem Cell Pluripotency*

“…However, in rESCs, the 2i culture condition induces a surprisingly high amount of Cdx2, a trophectoderm/TSC-specific marker (4,9). Because TSCs contribute to the placental development rather than the embryo proper, a strong expression of Cdx2 could further inhibit the developmental potency of rESCs.…”

“…Both 2i and 2i/LIF culture conditions also efficiently maintain pluripotency in mESCs (8). However, unlike mESCs, rESCs that are established and maintained in 2i and 2i/LIF express relatively high levels of trophoblast stem cell (TSC)-specific factors, like CDX2 (4,9), and have a propensity for genomic instability (2). Curiously, rESCs grown with Rhoassociated kinase and TGF-␤ inhibitors also showed much higher levels of CDX2 expression (9).…”

“…However, unlike mESCs, rESCs that are established and maintained in 2i and 2i/LIF express relatively high levels of trophoblast stem cell (TSC)-specific factors, like CDX2 (4,9), and have a propensity for genomic instability (2). Curiously, rESCs grown with Rhoassociated kinase and TGF-␤ inhibitors also showed much higher levels of CDX2 expression (9). Also, the efficiency of germ line competence in 2i-or 2i/LIF-cultured rESCs is lower than that of mESC and random.…”

Transcriptome Characteristics and X-Chromosome Inactivation Status in Cultured Rat Pluripotent Stem Cells

“…It was described that use of a GSK3 inhibitor, CHIR99021, in cultivation of rat and mouse ESCs caused ectopic expression of Cdx2 [33][34][35] encoding a wellknown trophoectodermal transcription factor [36]. According to RT-PCR and transcriptome analysis, Cdx2 was not expressed in the rEF lines and was highly expressed (on the average, 1,192 normalized read counts) in the rat pluripotent cells (Fig.…”