Wnt signaling blockade is essential for maintaining the pluripotency of chicken embryonic stem cells.

To determine whether the chESCs maintained pluripotency under 4i + LIF conditions, the expression levels of pluripotency-related ( NANOG, SOX2, SOX3, OCT4, PRDM14, DNMT3B , and LIN28A ) and germ cell ( CVH and DAZL ) markers were analyzed; the expression levels of some these markers showed an increasing trend over time from d 5 to 9 ( Figure 3 C).

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When the glycogen synthase kinase-3 β inhibitor CHIR99021—which activates Wnt signaling—was added to chESC cultures, the colony shape flattened, and the expression levels of pluripotency-related ( NANOG, SOX2, SOX3, OCT4, LIN28A, DNMT3B , and PRDM14 ) and germ cell ( CVH and DAZL ) markers showed a decreasing trend, and the growth of chESCs was inhibited after approximately 7 d.

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