Research ArticleDEVELOPMENTAL BIOLOGY

Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaax7525
DOI: 10.1126/sciadv.aax7525
  • Fig. 1 The CDP-Etn pathway is required for efficient somatic cell reprogramming.

    (A) Lipid profiles of MEFs, mESCs, and MEFs undergoing Sox2, Klf4, and Oct4 (SKO) reprogramming on days 2, 4, 6, and 8. n = 6. Lipid species identified are listed in table S1. (B) Schematic of key phospholipid synthesis pathways. (C) qRT-PCR analysis of expression of genes encoding rate-limiting enzymes in phospholipid synthesis pathways on days 0, 2, and 4 in MEFs transduced with SKO in iCD1 medium. Data are represented as mean ± SD (n = 3). **P < 0.01 and ***P < 0.001. (D) Cellular PE levels were measured in MEFs transduced with SKO in iCD1 medium on days 0, 2, and 4. Data are represented as mean ± SD (n = 7). **P < 0.01 and ***P < 0.001. (E) Knockdown of Etnk1 and Etnk2 (shEtnk1/2) or Pcyt2 (shPcyt2) impaired reprogramming efficiency. The numbers of Oct4-GFP+ colonies were counted on day 6. shRNA against luciferase (shLuc) was used as control. Data are represented as mean ± SD (n = 4). ***P < 0.001. (F) The numbers of Oct4-GFP+ colonies were counted on day 6 in MEFs transduced with SKO in iCD1 medium or iCD1 medium without Etn (ΔiCD1). Data are represented as mean ± SD (n = 4). ***P < 0.001. (G) The numbers of Oct4-GFP+ colonies were counted on day 6 in MEFs transduced with SKO in ΔiCD1 medium supplemented with vehicle, Etn, CDP-Etn, or L-α-PE. Data are represented as mean ± SD (n = 3). *P < 0.05 and ***P < 0.001. (H) Etn had no effect on reprogramming when Etnk1/2 or Pcyt2 was silenced by shRNAs. The numbers of Oct4-GFP+ colonies were counted on day 6. shLuc was used as control. Data are represented as mean ± SD (n = 3). ***P < 0.001. (I) Suppression of the CDP-Etn pathway by shRNA against Etnk1/2 or Pcyt2 or Etn deprivation had no effect on the proliferation of MEFs undergoing reprogramming. Data are represented as mean ± SD (n = 3). ns, not significant. (J) Etn deprivation inhibited ESC growth. Representative phase-contrast and Oct4-GFP images (left) and growth curves (right) of ESCs in complete medium (control) or medium deprived of Etn. Scale bar, 250 μm. Data are represented as mean ± SD (n = 3). *P < 0.05. (K) Knockout (KO) of Pcyt2 significantly impaired ESC growth. Representative phase-contrast and Oct4-GFP images of wild-type (WT) and Pcyt2 knockout ESCs. Scale bar, 100 μm.

  • Fig. 2 The CDP-Etn pathway accelerates MET during reprogramming.

    (A) Experimental design of RNA-seq for profiling of genes regulated by the CDP-Etn pathway during reprogramming. shLuc was used as control. (B) Venn diagram showing the substantial overlap of Etn-, shEtnk1/2-, and shPcyt2-regulated genes on day 2 of SKO reprogramming. (C) GO analysis of genes regulated by the CDP-Etn pathway on day 2 of SKO reprogramming. (D) Heatmap showing the expression of selected epithelial genes (Cdh1, Epcam, Krt7, Krt8, Ocln, and Tjp2) and mesenchymal genes (Snail, Twist1, Twist2, Zeb1, Vim, Itga7, Col1a1, Col1a2, Col5a1, and Col5a2) on day 2 of SKO reprogramming. (E) qRT-PCR analysis of expression of selected epithelial and mesenchymal genes on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium (left) and in MEFs transduced with SKO in ΔiCD1 or iCD1 medium (right). Data are represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Western blot analysis (left) and corresponding quantification (right) of Cdh1 expression on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium or in MEFs transduced with SKO in ΔiCD1 or iCD1 medium. Data are represented as mean ± SD (n = 3). **P < 0.01 and ***P < 0.001. (G and H) Representative images (G) and quantification (H) of scratch assays on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium (left) or in MEFs transduced with SKO in ΔiCD1 or iCD1 medium (right). Scale bar, 100 μm. Data are represented as mean ± SEM (n = 4). **P < 0.01 and ***P < 0.001. (I) Flow cytometry analysis of Cdh1 in MEFs and MEFs transduced with SKO in ΔiCD1 or iCD1 medium. Representative images of immunofluorescence staining of Cdh1 (green) are shown. Scale bar, 100 μm. (J) Etn had no effect on reprogramming in the presence of TGF-β1, TGF-β2, or TGF-β3. The numbers of Oct4-GFP+ colonies were counted on day 6. Data are represented as mean ± SD (n = 3). ***P < 0.001.

  • Fig. 3 The CDP-Etn pathway acceleration of MET depends on Pebp1.

    (A) qRT-PCR analysis (left) and Western blot analysis (right) of expression of Pebp1 on days 0, 2, 4, and 6 in MEFs transduced with SKO in iCD1 medium. Data are represented as mean ± SD (n = 3). **P < 0.01 and ***P < 0.001. (B) Knockdown of Pebp1 (shPebp1) impaired reprogramming efficiency. The numbers of Oct4-GFP+ colonies were counted on day 6. Data are represented as mean ± SD (n = 3). ***P < 0.001. (C) Venn diagram showing the overlap of genes regulated by the CDP-Etn pathway and Pebp1 on day 2 of SKO reprogramming. (D) GO analysis of genes regulated by both the CDP-Etn pathway and Pebp1 on day 2 of SKO reprogramming. (E) qRT-PCR analysis of expression of selected epithelial and mesenchymal genes on day 2 in MEFs transduced with SKO and shLuc or shPebp1 in iCD1 medium. Data are represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Flow cytometry analysis of Cdh1 in MEFs and MEFs transduced with SKO and shLuc or shPebp1 in iCD1 medium. Representative images of immunofluorescence staining of Cdh1 (green) are shown. Scale bar, 100 μm. (G and H) No significant differences in cell migration were observed between reprogramming in ΔiCD1 or iCD1 medium when Pebp1 was knocked down. Representative images (G) and quantification (H) of scratch assays were analyzed on day 2. Scale bar, 100 μm. Data are represented as mean ± SEM (n = 4). **P < 0.01. (I) No significant differences in reprogramming efficiency were observed between reprogramming in ΔiCD1 or iCD1 medium when Pebp1 was knocked down. The numbers of Oct4-GFP+ colonies were counted on day 6. Data are represented as mean ± SD (n = 3). ***P < 0.001.

  • Fig. 4 The CDP-Etn-Pebp1 axis modulates NF-κB signaling to inhibit mesenchymal genes.

    (A) Western blot analysis of NF-κB p65 in the nuclear fraction and whole cell on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, shPcyt2, or shPebp1 in iCD1 medium (left) or in MEFs transduced with SKO and shLuc or shPebp1 in ΔiCD1 or iCD1 medium (right). (B) Coimmunoprecipitation analysis of the interaction between Pebp1 and IKKα or IKKβ on day 2 in MEFs undergoing reprogramming in the absence or presence of Etn. (C) Western blot analysis of phosphorylation of IKKα/β on day 2 in MEFs transduced with SKO and shLuc or shPebp1 in the absence or presence of Etn. (D) ChIP-qPCR analysis of NF-κB binding at the Snail (left) and Twist1 (right) promoters on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, shPcyt2, or shPebp1 in iCD1 medium. Schematic of primer sets for ChIP-qPCR on the Snail and Twist1 promoters is shown above. Data are represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. (E) Western blot analysis of Snail and Twist1 expression on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, shPcyt2, or shPebp1 in iCD1 medium. (F) ChIP-qPCR analysis of NF-κB binding at the Snail (left) and Twist1 (right) promoters on day 2 in MEFs transduced with SKO and shLuc or shPebp1 in ΔiCD1 or iCD1 medium. Data are represented as mean ± SD (n = 3). ***P < 0.001. (G) Western blot analysis of Snail and Twist1 expression on day 2 in MEFs transduced with SKO and shLuc or shPebp1 in ΔiCD1 or iCD1 medium. (H) Proposed model for the CDP-Etn pathway in pluripotency acquisition.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax7525/DC1

    Fig. S1. The CDP-Etn pathway is required for somatic cell reprogramming.

    Fig. S2. Characterization of iPSCs generated from iCD1 or ΔiCD1 and generation of Pcyt2 knockout mESCs.

    Fig. S3. The CDP-Etn pathway acceleration of MET depends on Pebp1.

    Fig. S4. The CDP-Etn-Pebp1 axis modulates NF-κB signaling to inhibit mesenchymal genes.

    Table S1. Lipid species identified MEFs, mESCs, and MEFs undergoing SKO reprogramming on days 2, 4, 6, and 8.

    Table S2. shRNA target sequences.

    Table S3. Primers for qRT-PCR.

    Table S4. Primers for ChIP-qPCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The CDP-Etn pathway is required for somatic cell reprogramming.
    • Fig. S2. Characterization of iPSCs generated from iCD1 or ΔiCD1 and generation of Pcyt2 knockout mESCs.
    • Fig. S3. The CDP-Etn pathway acceleration of MET depends on Pebp1.
    • Fig. S4. The CDP-Etn-Pebp1 axis modulates NF-κB signaling to inhibit mesenchymal genes.
    • Table S1. Lipid species identified MEFs, mESCs, and MEFs undergoing SKO reprogramming on days 2, 4, 6, and 8.
    • Table S2. shRNA target sequences.
    • Table S3. Primers for qRT-PCR.
    • Table S4. Primers for ChIP-qPCR.

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