Expression and functions of FOXM1 in human embryonic stem cells

Human embryonic stem cells (hESCs) are characterized by unlimited proliferation (self-renewal), capability to differentiate into derivatives of all three germ layers (pluripotency), and abbreviated cell cycle structure. Despite tremendous efforts in identification of important regulators, the complicated molecular mechanisms and essential effectors underlying the distinctive features of hESCs have not yet been fully elucidated. Forkhead box transcription factor M1 (FOXM1) has been demonstrated to be critical for the maintenance of pluripotency in mouse embryonic stem cells (mESCs) and mouse embryonal carcinoma cells (mECCs). The present study hypothesized that FOXM1 is important to the self-renewing capacity and pluripotency of hESCs. The objectives of this study were to characterize FOXM1 expression in undifferentiated and differentiating hESCs, and to study the effect of perturbing FOXM1 expression on pluripotency and proliferation. Undifferentiated VAL3 analyzed by bivariate flow cytometric analysis revealed that FOXM1 expression was regulated in a cell cycle phase-dependent manner, with expression level increased from G1 through S phase, and eventually reached peak levels in G2/M phase. To study the subcellular localization of FOXM1 with respect to cell cycle progression, VAL3 cells were synchronized by nocodazole-mediated cell cycle block, followed by immunocytochemical analysis. The result indicated that FOXM1 underwent nuclear translocation at late-S and early-G2 phase of the cell cycle. When VAL3 spontaneously differentiated as embryoid bodies (EBs), the mRNA expression of FOXM1 displayed profound fluctuation over the differentiation process. Retinoic acid (RA) treatment induced rapid differentiation of VAL3, yet differential expression pattern of FOXM1 was observed for cells grown in different culture media. FOXM1 mRNA expression persisted in differentiating VAL3 cultured in mTeSR. By contrast, RA-driven differentiation of VAL3 cultured in conditioned medium was accompanied by transient depletion and resurgence of FOXM1 protein expression. Differentiation of VAL3 driven by Definitive Endoderm kit did not alter FOXM1 expression, whereas induced differentiation by Bone Morphogenic Protein 4 (BMP4) led to repression of FOXM1. The functional role of FOXM1 in hESCs was investigated with the use of siRNA. Transient knockdown of FOXM1in VAL3 did not induce substantial repression of pluripotent marker (OCT4, SOX2, NANOG) expression nor significant morphological change of colonies, despite upregulation of early differentiation marker SSEA-1. Intriguingly, FOXM1 depletion led to altered cell cycle progression and delay in G2 phase progression, possibly attributed to the downregulation of Cyclin B1 and Cdc25B. Also FOXM1 knockdown impaired VAL3 proliferation, yet no prominent defect in mitosis was observed. In conclusion, the present study reported for the first time the expression and functions of FOXM1 in undifferentiated hESCs. Upon differentiation, FOXM1 expression varied in cells committing to different lineages. Depletion of FOXM1 did not interfere with hESCs pluripotency, but hindered cell cycle progression and cell proliferation, suggesting that FOXM1 is mainly involved in promoting rapid proliferation of hESCs. The functional role and regulatory mechanics of FOXM1 in hESCs cell cycle control and differentiation warrant further investigation.published_or_final_versionBiochemistryMasterMaster of Philosoph