Metabolic switch and epithelial–mesenchymal transition cooperate to regulate pluripotency

Abstract Both metabolic switch from oxidative phosphorylation to glycolysis (OGS) and epithelial–mesenchymal transition (EMT) promote cellular reprogramming at early stages. However, their connections have not been elucidated. Here, when a chemically defined medium was used to induce early EMT during mouse reprogramming, a facilitated OGS was also observed at the same time. Additional investigations suggested that the two events formed a positive feedback loop via transcriptional activation, cooperated to upregulate epigenetic factors such as Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5, and accelerated pluripotency induction at the early stage. However, at late stages, by over‐inducing glycolysis and preventing the necessary mesenchymal–epithelial transition, the two events trapped the cells at a new pluripotency state between naïve and primed states and inhibited further reprogramming toward the naïve state. In addition, the pluripotent stem cells at the new state have high similarity to epiblasts from E4.5 and E5.5 embryos, and have distinct characteristics from the previously reported epiblast‐like or formative states. Therefore, the time‐dependent cooperation between OGS and EMT in regulating pluripotency should extend our understanding of related fields.

(B) The protein levels of NANOG and REX1 were determined in two 5C-Oct4GFP + colonies with immunofluorescence. 5C-Oct4GFP-colonies have no detectable expression of these factors. Scale bar, 50 μM.
(C) The DNA methylation on the promoters of Nanog and Oct4 were determined in 5C-GFP + cells with bisulfate sequencing.
Data information: Experiments were independently repeated at least five times (n≥5). Error bars represent standard deviations. Additional statistical information was listed in Dataset EV7.

Appendix Figure S2
Energy metabolism is modulated by different methods (related to Figure 1) (A) 5C or mES medium was used during reprogramming. On day 6, the expression of several glycolysis markers was determined with qPCR and normalized against those in MEFs.
(B) Reporter for HIF1α activity was delivered into MEFs 24 hours before reprogramming via a lentivirus system as described in Materials and Methods. The YFP fluorescence was determined at the beginning of reprogramming (MEFs or day 0) as control with FACS. The YFP fluorescence activity was also determined on day 3 and 6 during reprogramming with mES or 5C medium.
(C) The abilities of sh-RNAs (sh-Hif1α, sh-Pdk1, and sh-Pdk2) to suppress the expression of target genes were determined three days after being delivered into MEFs. The expression of target genes in cells with tested shRNAs were normalized against and compared with that in cells with sh-Luc.
(D-E) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system. Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in ECAR after adding glucose was considered as glycolysis ability of the cells, while decrease in OCR after adding oligomycin was considered as the ATP production ability of the cells.
(F-G) Energy metabolism was controlled by modulating the expression of Pdk1/2 with a retrovirus system or using small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM). Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Glycolysis ability and ATP production ability of the cells were listed.
Data information: Experiments were independently repeated at least five times (n≥5) except Pscan analysis. Error bars represent standard deviations. ***p < 0.001. Additional statistical information was listed in Dataset EV7.

Appendix Figure S3
5C medium facilitates the reprogramming by reducing pre-iPSCs formation (related to Figure 2 Genes with significant expression differences (Log2 value change over 1.5) between pre-iPSCs and iPSCs/ESCs were selected (heatmap on left). The expression of these genes in pre-iPSCs, iPSCs, and ESCs were normalized against those in MEFs and plotted in heatmap in middle. Similarly, after normalizing against corresponding gene expression in MEFs, the expression of these genes in 5C-Oct4GFPand mES-Oct4GFPcells were listed and plotted (current RNA-Seq, heatmap on right).
(B) Four types of expression barriers during the conversion from pre-iPSCs to iPSCs were summarized.
The genes which were required to be down-or up-regulated during reprogramming (from MEFs to iPSCs) but were not down-or up-regulated in pre-iPSCs, were considered as type I or II barriers. The genes which expressed at similar levels in MEFs and iPSCs but were significantly up-or down-regulated in pre-iPSCs, were considered as type III or IV barriers. The numbers of barriers that have already been overcome or not identified in 5C-Oct4GFPand mES-Oct4GFPwere summarized.
(C) The expression of typical methyltransferases and demethylases targeting H3K9 were determined in 5C-Oct4GFPand mES-Oct4GFPcells with qPCR on day 6 during reprogramming.
(D) The abilities of sh-RNAs (sh-Bmi1, sh-Ctcf, sh-Ezh2, sh-Kdm2b and sh-Wdr5) to suppress the expression of target genes were determined on day 6 during reprogramming with 5C medium. The expression of target genes in cells with tested shRNAs were normalized against and compared with that in cells with sh-Luc.
(E) The abilities of sh-RNAs (sh-Snai2, sh-Twist1, sh-Twist2, and sh-Zeb1) to suppress the expression of target genes were determined three days after being delivered into MEFs. The expression of target genes in cells with tested shRNAs were normalized against and compared with that in cells with sh-Luc.

Appendix Figure S4
Early EMT and OGS during other cell fate conversions (related to Figure 4 Data information: Additional statistical information was listed in Dataset EV7.

Appendix Figure S5
Metabolic state contributes to reprogramming directly (related to Figure 4) (A-B) Hif1α was overexpressed via a retrovirus system during reprogramming with mES medium.
Oligomycin (1 µM) was used in another group. The expression of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined on day 6 with qPCR (A). The expression of Hif1α, Pdk1, and Pdk2 was determined on day 6 with qPCR (B).
(C) The expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system. Energy metabolism was directly controlled by using small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM). These different treatments were combined during reprogramming with mES medium. The Oct4GFP + colonies were counted on day 15 and normalized against the number in group without any treatment.
(D-E) Early EMT and the expression of related transcriptional factors were regulated by using TGFβ (TGFβ1/2/3, 1ng/ml each) and Repsox (1 μM) on day 2-7. Experiments in (C) were repeated in the context of TGFβ (D) and Repsox treatment (E). The Oct4GFP + colonies were counted on day 15 and normalized against the number in group without any treatment (heatmaps on the left). Addition normalization against the corresponding results in (C) was performed and the final results were listed in heatmaps on the right to make the functions of TGFβ and Repsox clearer.
(F) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system. Energy metabolism was directly controlled by using small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM). These different treatments were combined during reprogramming with mES medium. The Oct4GFP + colonies were counted on day 15 and normalized against the number in group without any treatment.

Appendix Figure S6
Early EMT and OGS are also observed during other cell fate conversions (related to Figure 5) (A-B) The expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system. Energy metabolism was directly controlled by using small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM). The Oct4GFP + colonies were counted on day 15 during reprogramming with mES (A) or 5C medium (B). The numbers of three types of Oct4GFP + colonies were summarized.
The numbers of three types of Oct4GFP + colonies on day 15 were summarized.
Data information: Experiments were independently repeated at least five times (n≥5). Error bars represent standard deviations. Additional statistical information was listed in Dataset EV7.