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. 2022 Mar;18(3):1181-1192.
doi: 10.1007/s12015-021-10303-x. Epub 2021 Nov 20.

Nox4-IGF2 Axis Promotes Differentiation of Embryoid Body Cells Into Derivatives of the Three Embryonic Germ Layers

Jusong Kim #  1 , Jaewon Kim #  1   2 , Hee Jung Lim  1 , Sanghyuk Lee  1   2 , Yun Soo Bae  3 , Jaesang Kim  4   5
Affiliations

Nox4-IGF2 Axis Promotes Differentiation of Embryoid Body Cells Into Derivatives of the Three Embryonic Germ Layers

Jusong Kim et al. Stem Cell Rev Rep. 2022 Mar.

Abstract

Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4-/-) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4-/- cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4-/- iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells.

Keywords: ES cells; Embryoid body; IGF2; Nox4; ROS; iPSCs.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1
ROS is required for efficient differentiation of EB. (A) EBs cultured in differentiation media for 7 days with or without addition of 250 nM DPI and Coomassie blue-stained. Scale bar: 500 μm. Graph on the right side shows diameters of multiple EB colonies from ± DPI cultures with a significant difference (***P-value of <0.0005 from Student’s t-test). (B) Graph summarizing results from flow cytometric analyses for apoptosis following treatment with DPI for 24 and 48 h. Early apoptosis indicates % Annexin V+ cells, and late apoptosis indicates % PI+ Annexin V+ cells. Note that no significant differences were seen between treated and untreated controls (‘n.s.’ indicates ‘not significant’ from Student’s t-test). (C) Representative images of immunofluorescence staining showing expression of representative markers of the three embryonic germ layers. βIII-tubulin, SMA, and α-fetoprotein are used as markers for ectoderm, mesoderm, and endoderm respectively. Cells from EBs are cultured with or without 250 nM DPI for 7 days and counterstained with DAPI (blue). Scale bar: 50 μm. (D) Real time RT-PCR analyses showing changes in representative marker gene expression during EB differentiation. 5 markers for each of the germ layers are examined on days 0, 7 and 14. Error bars represent mean ± S.D. from five independent experiments. Statistical significance is indicated (*P-value of <0.05, ** P-value of <0.005, *** P-value of <0.0005 from Student’s t-test)
Fig. 2
Fig. 2
Expression of Nox4 during EB differentiation. (A) Expression of Nox isozyme genes during differentiation. Real time RT-PCR analyses on samples from indicated days show only Nox4 is up-regulated peaking on day 7 (**P-value of <0.005 from Student’s t-test). (B) Immunoblot showing changes in Nox4 expression. α-tubulin is used as the loading control. (C) Levels of ROS are examined by DCF-DA fluorescence. Histograms are color-coded for differently for days 0 (mouse embryonic stem cells: mES), 7 and 14 after initiation of differentiation. Graph to the right shows results from two independent flow cytometric analyses. Percentages of cells within the indicated range are shown. Statistical significance is indicated (*P-value of <0.05 from Student’s t-test)
Fig. 3
Fig. 3
Generation and characterization of Nox4−/− iPSCs. (A) Immunoblot showing the absence of Nox4 protein in mouse embryonic fibroblasts (MEF) isolated from Nox4−/− mouse. (B) Immunoblot showing expression of Yamanaka factors in iPSCs from wild type (WT) and Nox4−/− mice. MEF are used as negative controls. (C) Alkaline phosphatase activity in iPSCs from WT and Nox4−/− mice. (D) Immunostaining for expression of Oct4, Sox2 and SSEA-1 in iPSCs from WT and Nox4−/− mice. Scale bar: 50 μm. (E) Representative images of immunofluorescence staining showing expression of representative markers of the three embryonic germ layers. Cells from iPSC-derived EBs were cultured, immunostained and counterstained with DAPI (blue). Scale bar: 50 μm. (F) Levels of ROS are examined by DCF-DA fluorescence on days 7 and 14 after initiation of differentiation. Histograms are color-coded for different cells under examination. Graphs below show results from three independent flow cytometric analyses. Percentages of cells within the indicated range are shown. Statistical significance is also indicated (*P-value of <0.05 from Student’s t-test)
Fig. 4
Fig. 4
Time-course analysis of transcriptional changes in WT and Nox4−/− EB cells. (A) Venn diagram of DEGs (Differentially Expressed Genes) between wild-type and Nox4−/− cells determined by two analytical tools, edgeR and EBSeqHMM. 597 DEGs were selected as genes with significant (FDR <0.01 in edgeR and FDR <1e-10 in EBSeqHMM) patterns in WT compared to Nox4−/− cells. (B) Heatmap of log2 TPM (Transcripts Per Million) expression of 597 DEGs in WT and Nox4−/− cells. The heatmap was scaled to row-wise z-score. (C) Gene set over-representation analysis of KEGG pathways for 597 DEGs by ConsensusPathDB. Pathways with FDR <0.05 are shown. The full list and details are in the Supplementary Table 2. The number of DEGs in each pathway is indicated next to the bar. The dotted vertical line indicate P-value of 0.05. (D) Log2 TPM expression for growth factors among DEGs in the MAPK signaling pathway. Genes were sorted by FDR of edgeR shown in the bar plot to the right of the heatmap
Fig. 5
Fig. 5
Nox4 promotes differentiation of three embryonic germ layers via IGF2 induction. (A) Expression of IGF2 during differentiation of EB cells (*P-value of <0.05, ** P-value of <0.005, *** P-value of <0.0005 from Student’s t-test) from real time RT-PCR analyses. Note the reduced levels in Nox4−/− cells. (B) EBs cultured in differentiation media for 7 days with or without addition of IGF2 and Coomassie blue-stained. Graph on the right side shows diameters of multiple EB colonies from ± IGF2 cultures with significant difference (*P-value <0.05 from Student’s t-test) in the case of Nox4−/− EBs. (C) Representative images of immunofluorescence staining showing expression of representative markers of the three embryonic germ layers. Cells from Nox4−/− EBs are cultured with or without IGF2 for 7 days and counterstained with DAPI (blue). Scale bar: 50 μm
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