Recent reports have indicated the functional significance

Recent reports have indicated the functional significance of FOXM1 in pluripotent cells. FOXM1 is required for the maintenance of pluripotency in mouse embryonal carcinoma (EC) cells, via direct regulation of Oct4 transcription (Xie et al., 2010). More importantly, FOXM1 is important for maintaining pluripotency in mouse ES cells as a downstream target of the LIF/STAT3 signaling pathway, thereby stimulating the expression of pluripotent genes (Tan et al., 2014). Depleting FOXM1 in mouse ES cells led to a rapid loss of pluripotency as well as a decreased rate of cell proliferation (Tan et al., 2014). A recent study in human EC cells provided evidence that FOXM1 is required for OCT4 expression, underscoring the potential functional role of FOXM1 in the context of human pluripotent stem cells (Chen et al., 2015). However, a detailed study of the expression and function of FOXM1 in hESCs is still lacking.
Materials and methods
Results
Discussion FOXM1 has been extensively studied in cancer cells and during mouse development and shown to play a role in a broad spectrum of biological processes. The first link of FOXM1 to the regulation of stem cell pluripotency came with the demonstration that FOXM1 acted through the core pluripotent circuitry to mediate the maintenance of pluripotency and self-renewal in mouse pluripotent cells (Xie et al., 2010; Tan et al., 2014). This notion was further strengthened when FOXM1 knockdown was shown to downregulate OCT4 expression in human EC cells (Chen et al., 2015), but the functional relevance of FOXM1 in hESC remain unclear. In this study, we showed that FOXM1 was highly expressed in undifferentiated hESCs. Similar to other cell types (Korver et al., 1997b; Leung et al., 2001; Laoukili et al., 2008), FOXM1 displayed periodic expression during the BTS supplier with heightened levels at G2/M. FOXM1 levels did not decrease precipitously like the core pluripotent markers but showed fluctuations in spontaneously differentiating VAL-3 cells. Due to the random nature of spontaneous differentiation, EBs consist of a mix of cells committing to various lineages (Pekkanen-Mattila et al., 2010). The fluctuating expression of FOXM1 during EB differentiation suggests that FOXM1 expression/function may be required in certain lineage differentiation pathways but does not necessarily correlate with the undifferentiated state of hESCs. Indeed, in vitro differentiation of VAL-3 cells induced by various morphogens gave different outcomes. While downregulation was observed in differentiating hESCs upon RA and BMP4 induction, FOXM1 expression was sustained following differentiation driven by AVBF (mesodermal) and a definitive endoderm differentiation kit. The functional requirement of FOXM1 during differentiation into mesodermal and endodermal derivatives requires further investigation. Our findings do not support the direct regulation of pluripotent genes by FOXM1 during hESC differentiation, in contrast to previous findings in mouse and human EC cells (Xie et al., 2010; Chen et al., 2015). In line with the differentiation experiments, siRNA-mediated knockdown of FOXM1 in hESCs did not induce rapid loss of undifferentiated state or substantial downregulation of the core pluripotent markers. Our data differ from the rapid loss of the undifferentiated state and downregulation of OCT4 and NANOG upon FOXM1 knockdown in mouse pluripotent cells (Xie et al., 2010; Tan et al., 2014). FOXM1 binds to the OCT4 promoter, stimulating its expression in both mouse and human EC cells (Xie et al., 2010; Chen et al., 2015). Interestingly, we did observe subtle and transient downregulation of OCT4 and NANOG in VAL-3 cells two days after FOXM1 siRNA transfection, as well as induced expression of SSEA-1, indicating signs of differentiation upon FOXM1 knockdown. Further, enrichment of FOXM1 binding at the promoter of OCT4 suggests that FOXM1 might participate in the transcriptional regulation of OCT4. The subtle and transient effect observed in our study may be due to compensatory regulation upon FOXM1 knockdown. Interestingly, one of the top enriched binding motifs that coexists with FOXM1 binding is recognized by PRDM14, which was recently shown to be essential to pluripotency through interactions with PRC2 to establish repressive histone modifications and suppress differentiation genes in hESCs (Chia et al., 2010; Chan et al., 2013). Potential interactions between FOXM1 and PRDM14 may be specific to hESCs and highlight a possible link of FOXM1 to pluripotency through a mechanism other than direct transcriptional control of the core pluripotent genes.