Roles Of Transcription Factor Foxm1in Maintaining Mouse Embryonic Stem Cell Pluripotency And Induced Pluripotent Stem Cell Reprogramming

Embryonic stem cells(ESCs) are derived from the inner cell mass of mammalianblastocyst, have the unique ability to grow indefinitely in culture while retaining theirpluripotency and differentiate into derivatives of all three cmbryonic germ layers.Becasue of their plasticity and potentially unlimited capacity for self-renewal, ESCshave been proposed for gene function research, developmental biology, drug selection,regenerative medicine, and tissue replacement after injury or disease. The pluripotencyof ESCs are regulated by many extracellular signal factors, intracellular complexregulatory networks, and transcriptional networks, so it is meaningful to understandthe mechanism of how ESCs maintain their pluripotent cell identity. Foxm1is amember of the Fox family of trancription factors. Its expression and transcriptionalactivity are regulated by many intracellular factors and cell signal pathway, related tocell proliferation, aging, and embryogenesis. The abnormal upregulation of Foxm1isinvolved in tumor development and metastasis. Our previous studies have confirmedthat Foxm1participates in maintenance of pluripotency of mouse P19embryonalcarcinoma cells. Here we studied the role of Foxm1in maintaining the pluripotency ofmouse embryonic stem cell (mESC) line D3and in reprogramming the somatic cellsinto induced pluripotent stem cells.In conventional culture conditons of mESCs, LIF is a necessary cytokine tomaintain the pluripotency of mESCs. It binds to a heterodimeric receptor consisting ofthe LIF receptor and gp130, which phosphorylates Stat3by activating JAKs. T hephosphorylation of Stat3results in homodimerization of Stat3, which enters thenucleus and activates the transcription of Stat3downstream target genes. Withdrawalof LIF from mESCs medium resulted in the differentiation of the stem cells and thedecreased expression of Foxm1. Addition of LIF after two days withdrawal of LIFinducted the expression of Foxm1. ChIP and EMSA confirmed the specificity of Stat3binding activity on Foxm1promoter.To confirm the essential role of Foxm1in maintaining the pluripotency of mESCs,we constructed a lentiviral vector expressing Foxm1-specific shRNA. The Foxm1shRNA lentivirus was used to infect D3ES cells and the knockdown of Foxm1expression resulted in the loss of the typical mESC morphology, positive alkalinephosphatase staining, and nomal embryoid body formation ability of the cells.These observations suggested an essential role of Foxm1in maintenance of the pluripotencyof mESCs.We then constructed a FOXM1-overexpression lentiviral vector and used theFOXM1-overexpression lentivirus to infect D3ESCs. The FOXM1overexpressed D3ESCs maintained the typical mESC morphology and positive alkaline phosphatasestaining during a long term culture without LIF and feeders. Furthermore, thepluripotency of FOXM1overexpressing D3ESCs without LIF and feeders wasconfirmed by the nude mouse-grafted teratomas, in which contained derivatives of allthree germ layers. These observations suggested that the overexpression of FOXM1was sufficient to maintain the pluripotency of mESCs in the absence of LIF and feederlayers.To investigate the role of Foxm1in reprogramming mouse embryonic fibroblasts(MEFs) into induced pluripotent stem cells (iPSCs), we overexpressed FOXM1orknocked down the expression of Foxm1during the procedure of reprogramming. Wefound that overexpression of FOXM1has limited effects on the reprogramming whilethe knockdown of Foxm1abbolished the generation of iPSCs. The observationssuggested that Foxm1was essential for the reprogramming of somatic cells intopluripotent cells.In summary, we identified Foxm1as a critical LIF/Stat3downstream target thatmediated LIF/Stat3-dependent mESC self-renewal. Moreover, we found that theoverexpression of Foxm1alone maintained mESC pluripotency in the absence of LIFand feeder layers. In addition, we observed that the inhibition of Foxm1expressionabolished the reprogramming of MEFs to iPSCs. Together, our results revealed anessential function of Foxm1in the LIF/Stat3-mediated mESC self-renewal and thegeneration of iPSCs.