Foxm1 mediates LIF/Stat3-dependent self-renewal in mouse embryonic stem cells and is essential for the generation of induced pluripotent stem cells

Abstract

Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) is required for maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). Here, we have confirmed transcription factor Forkhead Box m1 (Foxm1) as a LIF/Stat3 downstream target that mediates LIF/Stat3-dependent mESC self-renewal. The expression of Foxm1 relies on LIF signaling and is stimulated by Stat3 directly in mESCs. The knockdown of Foxm1 results in the loss of mESC pluripotency in the presence of LIF, and the overexpression of Foxm1 alone maintains mESC pluripotency in the absence of LIF and feeder layers, indicating that Foxm1 is a mediator of LIF/Stat3-dependent maintenance of pluripotency in mESCs. Furthermore, the inhibition of Foxm1 expression prevents the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells (iPSCs), suggesting that Foxm1 is essential for the reprogramming of somatic cells into iPSCs. Our results reveal an essential function of Foxm1 in the LIF/Stat3-mediated mESC self-renewal and the generation of iPSCs.

Figure 1. LIF signaling pathways maintain the expression of Foxm1 in mESCs.

(A) D3 ES cells were cultured in the presence of feeders without LIF for one week. In the absence of LIF, D3 ES cells lost the typical colony morphology. The control D3 ES cells and D3 ES cells without LIF were cultured under exactly the same conditions for one week without replating. (B) Alkaline phosphatase staining and quantification of differentiated, mix, or undifferentiated colonies of D3 ES cells and D3 ES cells without LIF for one week. Error bars indicated standard deviation (n = 3). (C) The withdrawal of LIF resulted in the decreased levels of Foxm1 protein in D3 ES cells. Western blot analyses were performed for the expression of Foxm1, Klf4, Stat3, phosphorylated Stat3 (p-Stat3), and β-actin in D3 ES cells or D3 ES cells without LIF for two days. (D) Immunostaining of Foxm1 in D3 ES cells or D3 ES cells without LIF for one week in the presence of feeders. Pictures were taken with the UltraVIEW VoX Spinning Disk Confocal Microscope (Bar = 50 µm). (E) LIF withdrawal resulted in a rapid decrease of Foxm1 mRNA in D3 ES cells. Quantitative RT-PCR analyses were performed for Foxm1, Klf4, and Socs3 mRNA levels in D3 ES cells or D3 ES cells at 6 h, 12 h, and 24 h post LIF withdrawal. The mRNA levels of each transcript in D3 ES cells with LIF were set at 1.0. Error bars indicated standard deviation (n = 3).

Figure 2. Stat3 regulates the transcription of Foxm1.

(A) Response to the activation of LIF signaling. D3 ES cells were cultured in the presence of feeders without LIF for two days and then LIF was added back for another two days. Western blot analyses were performed with antibodies specific for Foxm1, Stat3, phosphorylated Stat3 (p-Stat3), and β-actin. (B) The response of Foxm1 promoter activity to LIF signaling. D3 ES cells were cultured in LIF-free and feeder-free conditions and transfected with a luciferase reporter plasmid containing −1.3 kb Foxm1 promoter and the pRL-CMV plasmid (loading control). LIF (1000 U/ml) was added 1 day later and luciferase activities were measured at 24 h after addition of LIF. (C) Sensitivity of LIF-mediated upregulation of Foxm1 to pathway-specific inhibitors. D3 ES cells were cultured without LIF for 4 days and then LIF was added for 6 h. The mix of two inhibitors (2i) of Mek (PD0325901 1 µM) and GSK3β (CHIR99021 3 µM), or JAK inhibitor I (10 µM) was added 1 hour before the addition of LIF. The mRNA levels of Foxm1, Klf4 and Socs3 were estimated by qPCR. (D–E) Stat3 bound to endogenous Foxm1 promoter. The position of a putative Stat3 binding site (TTCCNGGAA) in −2 kb mouse Foxm1 promoter was predicted by gene sequence analysis and the product of ChIP assays was also predicted. The chromatin of D3 ES cells was cross-linked, sonicated, and then immunoprecipitated (IP) with either Stat3 antibody or IgG (control) and the amount of promoter DNA associated with the IP chromatin was estimated by PCR/agarose gel electrophoresis (D) or qPCR (E) with the primers specific to Foxm1 promoter region. The predicted size of the PCR product was 328 bp. (F) Stat3 bound to the Stat3 binding sequence in Foxm1 promoter. Protein lysates were prepared from Stat3-overexpressing D3 ES cells and used for EMSA with a FAM-labeled DNA probe synthesized from the mouse Foxm1 promoter sequence position −1218 bp to −1192 bp. An unlabeled probe (100×), an unlabeled mutated probe (100×), or a Stat3-specific antibody was used in EMSA as controls. Protein lysates prepared from D3 ES cells without LIF and feeder layers was used as a control of the decreased Stat3 proteins.

Figure 3. Foxm1 is essential for maintaining the pluripotency of mESCs.

(A) The knockdown of Foxm1 expression resulted in the loss of the typical mouse ES cell morphology in D3 ES cells. A lentiviral vector expressing Foxm1-specific shRNA or a control shRNA was produced to infect D3 ES cells for two hours. The lentivirus-infected D3 ES cells were then cultured for one week with the standard ES cell culture condition. Pictures were taken at 200× magnification using a TE2000 microscope (Nikon). (B) The Foxm1 knockdown resulted in the decreased levels of Oct4 and Nanog in D3 ES cells. Western blot analyses were performed for the expression of Foxm1, Stat3, phosphorylated Stat3 (p-Stat3), Oct4, Nanog, GFP, and β-actin in D3 ES cells, D3 ES cells infected with the control lentivirus (NC shRNA), or D3 ES cells infected with the Foxm1 shRNA lentivirus (Foxm1 shRNA). (C) D3 ES cells infected with the Foxm1 shRNA lentivirus (Foxm1 shRNA) lost the positive alkaline phosphatase staining. The percentage of differentiated, mix, or undifferentiated colonies of D3 ES cells, D3 NC shRNA cells, or D3 Foxm1 shRNA cells was calculated. (D) The effects of Foxm1 knockdown on the mRNA levels of pluripotency-related genes in D3 ES cells. Quantitative RT-PCR analyses were performed for Foxm1, Utf1, Oct4, Nanog, Esrrb, Klf4, Tbx3, Klf2, and Sall4 mRNA levels in D3 ES cells, D3 NC shRNA cells, or D3 Foxm1 shRNA cells. The levels of each transcript in D3 ES cells were set at 1.0. Error bars indicated standard deviation (n = 3).

Figure 4. Overexpression of FOXM1 is sufficient to maintain the pluripotency of mESCs in the absence of LIF and feeder layers.

(A) The upregulation of FOXM1 expression maintained the typical mESC morphology and positive alkaline phosphatase staining in D3 ES cells in the absence of LIF and feeder layers. A lentiviral vector expressing human FOXM1B was produced to infect D3 ES cells and the lentivirus-infected D3 ES cells were cultured for one week in the absence of LIF and feeder layers. Pictures were taken at 200× magnification using a TE2000 microscope (Nikon). Alkaline phosphatase staining (AP) was performed in D3 ES cells, D3 ES cells or FOXM1-overexpressing D3 ES cells cultured without LIF and feeders for one week. The percentage of differentiated, mix, or undifferentiated colonies of different D3 ES cell samples was calculated. (B) The FOXM1 upregulation recovered the levels of Oct4 and Nanog in D3 ES cells in the absence of LIF and feeder layers. Western blot analyses were performed for the expression of Foxm1 (FOXM1), Stat3, phosphorylated Stat3 (p-Stat3), Oct4, Nanog, GFP, and β-actin in D3 ES cells, D3 ES cells without LIF and feeders for one week, or FOXM1-overexpressing D3 ES cells without LIF and feeders for one week. (C) The effects of FOXM1 overexpression on the mRNA levels of pluripotency-related genes in D3 ES cells without LIF and feeders at passage 5. Quantitative RT-PCR analyses were performed for Foxm1, Utf1, Oct4, Nanog, Esrrb, Klf4, Tbx3, Klf2 and Sall4 mRNA levels. The levels of each transcript in D3 ES cells were set at 1.0. Error bars indicated standard deviation (n = 3). (D) The typical mESC morphology and positive alkaline phosphatase staining were maintained in FOXM1 overexpressing D3 ES cells during long-term culture without LIF and feeders. Pictures were taken for FOXM1 overexpressing D3 cell culture without LIF and feeders at passage 5, passage 7, and passage 9. The colonies of typical positive alkaline phosphatase staining were shown in the squares of each picture. (E) The teratomas formed by FOXM1 overexpressing D3 ES cells without LIF and feeders at passage 5 contained derivatives of all three germ layers. Sections of the formed teratomas were stained with hematoxylin and eosin dyes and the representative photographs were taken using a TE2000 microscope.

Figure 5. Foxm1 is essential for the reprogramming of somatic cells into pluripotent cells.

(A) The procedure of iPCS induction with the infection of a cocktail of lentiviral vectors. (B) The knockdown of Foxm1 expression prevented the formation of iPSCs. MEFs (3×10⁴ cells) were infected by four lentiviruses that expressed Oct4, Sox2, Klf4, and c-Myc respectively (OSKM). The lentiviral vector expressing Foxm1-specific shRNA was coupled with the infection of OSKM four factors during iPSC formation. Fluorescent and light microscopy images were taken at day 10 of the iPSC formation. (C) Alkaline phosphatase staining of MEFs infected by OSKM or the four factors coupled with the Foxm1 shRNA lentivirus was performed at day 14 post lentiviral infection and the alkaline phosphatase positive (AP⁺) colony numbers were counted.