Research ArticleCELL BIOLOGY

Transient activation of the UPRER is an essential step in the acquisition of pluripotency during reprogramming

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Science Advances  10 Apr 2019:
Vol. 5, no. 4, eaaw0025
DOI: 10.1126/sciadv.aaw0025
  • Fig. 1 The three major unfolded protein responses are activated during cellular reprogramming.

    (A) Relative mRNA levels of the main effectors of the UPRER (HSPA5 and GRP94), HSR (HSPA1A), and UPRmt (GRP75) relative to GAPDH determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 3, average ± SD). GFP control was set to 1 for each day. (B) Western blot analysis of the main effectors of the UPRER (HSPA5 and GRP94), HSR (HSPA1A), and UPRmt (GRP75). D, day. (C) Time course reprogramming Western blot analysis of P-IRE1 and IRE1 with P-IRE1 quantification (n = 3, average ± SD). *P < 0.05, statistical difference using a Sidak multiple comparison test; n.s., statistical nonsignificance. (D) Relative mRNA levels of the spliced form of XBP1 relative to GAPDH determined by qRT-PCR (n = 3, average ± SD). GFP control was set to 1 for each day. (E) Electron microscopy of day 4 reprogramming fibroblasts and GFP control. Scale bar, 0.2 μm. Pseudocolors blue and red mark, respectively, the nucleus and the ER. (F) Secretion capability of the ER measured by luciferase activity secreted in the media (n = 12, average ± SD) and Western blot analysis of the Gaussia luciferase. (G) Sensitivity to tunicamycin treatment determined by median effective concentration (EC50) measurement at day 4 of reprogramming of fibroblast-like cells (n = 3, average ± SD). *P < 0.05, statistical difference using an unpaired two-tailed t test.

  • Fig. 2 Advanced state of reprogramming positively correlates with higher UPRERactivation.

    (A) Schematic of the genome editing strategy and Southern blot using a GFP probe. The red arrow shows the expected size of the targeted allele, while the black arrows show two off-target integrations. WT, wild type. (B) Schematic of the fibroblast-like cell differentiation protocol (left) and Western blot of HSPA5, GFP, and actin showing the dynamical induction of the reporter line after the addition of tunicamycin (0.1 μg/ml). The predicted HSPA5-GFP fusion band was targeted by both GFP and HSPA5 antibodies using dual-channel imaging with the Odyssey CLx Imaging System confirming the correct targeting. Only a single intense specific GFP band was observed, suggesting that the off-target integrations are not translated (right). (C) Median HSPA5-GFP levels analyzed by flow cytometry upon tunicamycin (0.1 μg/ml) treatment for 24 hours and after removal (n = 3, average ± SD). *P < 0.05, statistical difference using Dunnett’s multiple comparison test to the DMSO control. (D) Flow cytometry analysis of fibroblast-like HSPA5-GFP cells at day 8 of reprogramming stained with SSEA-4 and TRA-1-60 surface markers. I, II, and III represent the different cell states of reprogramming. (E) Median HSPA5-GFP of the different cell states (I, II, and III) during reprogramming (n = 3, average ± SD). *P < 0.05, statistical difference using Newman-Keuls multiple comparison test between all the conditions.

  • Fig. 3 Ectopic activation of the UPRERincreases the reprogramming efficiency.

    (A) Percentage of SSEA-4+/TRA-1-60+ cells at day 14 of reprogramming after drug treatment with APY29 (0.625 μM), an inducer of the UPRER, from day 4 to day 7 of reprogramming (n = 5, average ± SEM). *P < 0.05, statistical significant difference using an unpaired two-tailed t test. (B) Relative reprogramming efficiency of keratinocytes measured by colony TRA-1-60 staining after 3 weeks in culture upon overexpression of emGFP, XBP1s, and XBP1s-DBD (missing its DNA binding domain) with the EF1α promoter. Two biological replicates done in duplicate are shown (average ± SD). *P < 0.05, statistical difference using a Dunnett’s multiple comparison test to the control. (C) Relative reprogramming efficiency of keratinocytes measured by colony TRA-1-60 staining after 3 weeks in culture upon knockdown of XBP1 and ATF4 (n = 3, average ± SD). *P < 0.05, statistical difference using a Dunnett’s multiple comparison test to the control.

  • Fig. 4 Derived iPSCs express their endogenous pluripotent genes and are pluripotent.

    (A) Relative endogenous mRNA levels of pluripotent genes in the derived iPSC lines relative to GAPDH determined by qRT-PCR (n = 3, average ± SD). Values for H9 ESCs were set to 1. *P < 0.05, statistical difference using a Dunnett’s multiple comparison test to the control H9 ESC. (B) Hematoxylin and eosin staining of teratomas showing the three germ layers: mesoderm, ectoderm, and endoderm. Teratoma formation assays were performed after confirmation of the exogenous XBP1s silencing (see Fig. 5A). (C) Directed lineage-specific differentiation efficiencies assed by the percentage of cells expressing Brachyury (T) for mesoderm and SOX17 for endoderm differentiation by flow cytometry (n = 3, average ± SEM). n.s. indicates nonstatistical difference (P < 0.05) using a Dunnett’s multiple comparison test to the control H9 ESC.

  • Fig. 5 Transient activation of the UPRER is necessary during reprogramming.

    (A) Relative mRNA levels of XBP1s relative to GAPDH determined by qRT-PCR in iPSC colonies derived from either emGFP, XBP1s, or XBP1s-DBD driven by the EF1α promoter (n = 3, average ± SD). NB: This primer set will also recognize the XBP1s-DBD form. *P < 0.05, statistical difference using a Dunnett’s multiple comparison test to the control keratinoytes. (B) Flow cytometry analysis of HSPA5-GFP in ESC HSPA5-GFP and the differentiated fibroblast-like cells. FITC, fluorescein isothiocyanate. (C) Western blot analysis of ATF4, ATF6, and XBP1 in pluripotent stem cells and fibroblasts. Equal number of cells was loaded. (D) Relative mRNA levels of XBP1s and HSPA5 relative to GAPDH determined by qRT-PCR during the course of cellular reprogramming (n = 3, average ± SD). GFP control was set to 1 for each day. *P < 0.05, statistical significant difference using an unpaired two-tailed t-test.

  • Fig. 6 HSPA5-GFP levels predict the reprogramming efficiency.

    (A) Histogram of fibroblast-like HSPA5-GFP at day 8 of reprogramming. 1, 2, and 3 subdivide the population into three equal parts. Each of them is represented in the right panel by their SSEA-4 and TRA-1-60 staining. The percentage of double positive cells within each of these populations is shown. (B) Percentage of SSEA-4+/TRA-1-60+ cells within each population 1, 2, and 3 during reprogramming (n = 3, average ± SD). *P < 0.05, statistical difference using Newman-Keuls multiple comparison test between all the conditions for each day. (C) Upper panel shows relative reprogramming efficiency of fibroblast-like HSPA5-GFP sorted at day 7 of reprogramming based on their GFP levels and assessed by TRA-1-60 colony staining (n = 4, average ± SEM). Lower panel shows a representative picture of the staining. *P < 0.05, statistical difference using an unpaired two-tailed t test. (D) iPSC colony size distribution from the experiment in (C). The area of ~60 iPSC colonies was measured with ImageJ. There was no significant difference in the mean colony size (unpaired two-tailed t test). (E) Cells undergoing a successful cellular reprogramming activate the UPRER transiently (1). Increased levels of UPRER activation increase the efficiency of cellular reprogramming, (2) while decreasing the UPRER activation negatively affects the efficiency of cellular reprogramming (3). In our experimental design, it is possible to predict the efficiency of reprogramming based on the levels of UPRER activation around day 7, depicted by the predictability window. Cells unable to decrease the levels of UPRER activation give rise to iPSCs unable to properly spread (4).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaaw0025/DC1

    Fig. S1. The reprogramming factors activate the three major unfolded protein responses during reprogramming.

    Fig. S2. The reprogramming factors activate all the three branches of the UPRER during reprogramming.

    Fig. S3. Activation of the UPRER and reactivation of the endogenous pluripotent genes during the different cellular reprogramming stages using fibroblast-like HSPA5-GFP cells.

    Fig. S4. Modulation of the UPRER and its impact on cell proliferation.

    Fig. S5. Derived iPSCs stain positive for pluripotent genes.

    Fig. S6. Episomal reprogramming of fibroblasts by XBP1s overexpression.

    Fig. S7. Activation of the UPRER in stem cells prevents their proper spreading and cell sorting strategy.

    Fig. S8. Levels of UPRER activation are predictive of the reprogramming efficiency using 3F.

    Table S1. Transcriptome analysis of UPRER genes in fibroblasts, iPSCs, and ESCs.

    Table S2. List of reagents used.

    References (3436)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The reprogramming factors activate the three major unfolded protein responses during reprogramming.
    • Fig. S2. The reprogramming factors activate all the three branches of the UPRER during reprogramming.
    • Fig. S3. Activation of the UPRER and reactivation of the endogenous pluripotent genes during the different cellular reprogramming stages using fibroblast-like HSPA5-GFP cells.
    • Fig. S4. Modulation of the UPRER and its impact on cell proliferation.
    • Fig. S5. Derived iPSCs stain positive for pluripotent genes.
    • Fig. S6. Episomal reprogramming of fibroblasts by XBP1s overexpression.
    • Fig. S7. Activation of the UPRER in stem cells prevents their proper spreading and cell sorting strategy.
    • Fig. S8. Levels of UPRER activation are predictive of the reprogramming efficiency using 3F.
    • Table S1. Transcriptome analysis of UPRER genes in fibroblasts, iPSCs, and ESCs.
    • Table S2. List of reagents used.
    • References (3436)

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