Due to the capability of self-renewal and differentiation, embryonic stem cells (ESCs) are an invaluable tool for research in regenerative medicine. However, the main problem that hampers application of ESCs to practical stem cell therapies is that ESCs can grow as tumors when transplanted. To date, only one oncogene Eras is known to be responsible for tumorigenic growth of ESCs. Thus, the tumorigenic property of ESCs is poorly investigated. By global gene expression profiling of mouse ESCs, surprisingly I found that the oncogene Gli2, a downstream target of the growth factor Sonic hedgehog (Shh), was expressed in undifferentiated mouse ESCs, which was further validated by semi-quantitative RT-PCR and immunofluorescent microscopy. It is well-known that the Gli2 protein is activated by Shh. However, interestingly, the Gli2 transcripts were expressed abundantly in mouse ESCs without endogenous Shh expression detected.To test if expression of Gli2 in mouse ESCs is based on an autonomous mechanism or due to a signal(s) from the extracellular environment, mouse ESCs were cultured in either standard or chemically-defined serum-free conditions (CDSF) for 3 passages. Growth rates and expression levels of the transcription factor Oct3/4 (Pou5f1), a master regulator of pluripotency in mouse ESCs, were monitored. The mouse ESCs maintained in these conditions showed similar levels of Oct3/4 expression, but no endogenous Shh expression. However, intriguingly, the mouse ESCs maintained in CDSF conditions downregulated Eras and Gli2 expression, and exhibited a slower growth rate. Next, the mouse ESCs cultured in these conditions were injected subcutaneously into NOD-SCID mice to test their tumorigenicity. The mouse ESCs maintained in standard conditions generated a well-developed tumor in 4 weeks, whereas the mouse ESCs cultured in CDSF conditions failed to form a tumor up to 6 months. When CDSF culture was supplemented with the serum as a positive control, these mouse ESCs upregulated Gli2 expression, proliferated at the similar rate to the ones maintained in the normal conditions, and regained tumorigenicity. Remarkably, when mouse ESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, but had upregulated Gli2 expression. These findings indicate that Gli2 expression is tightly correlated with the incidence of tumor-like growth of mouse ESCs. Taken together, these results indicate that Gli2 expression is associated with the tumorigenic property of mouse ESCs, which can serve as a novel marker. Further, we suggest that tumorigenicity in mouse ESCs can be manipulated without comprising the pluripotency of mouse ESCs by maintaining mouse ESCs in CDSF conditions. The tumorigenic growth of mouse ESCs is reversed by pharmacological inhibition of Gsk3β, which suggests that Gsk3β governs the tumor-like growth of mouse ESCs by means of a mechanism different from the one to support the pluripotency of ESCs.
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