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. 2020 Apr 16;14(5):972–988. doi: 10.1016/j.stemcr.2020.03.017

Figure 3.

Figure 3

JAK-STAT3 Signaling Is Required for Primed to Naive Reprogramming and the Maintenance of Naive Pluripotency

(A) Protein abundance levels for cytokine receptors that can activate JAK-STAT3 signaling. Data show the mean ± SD of three (naive) or four (primed) biological replicates and were compared using a LIMMA-moderated t test with Benjamini-Hochberg correction (∗∗q < 0.01, ∗∗∗∗q < 0.0001).

(B) Western blot shows phosphorylated and total levels of STAT3 protein. Mass, kDa. Data in (B–E) were collected from naive H9 hPSC maintained in t2iLGö on Matrigel-coated plates and primed H9 hPSC maintained in TeSR-E8 on Vitronectin-coated plates. Results are representative of three biological replicates.

(C) Western blot confirms the decrease in phosphorylated STAT3 after JAK inhibition in naive hPSC. Mass, kDa.

(D) qRT-PCR analysis of gene expression levels in naive hPSC over a time course of JAK inhibition. Pluripotency-associated genes are shown in the top row and predicted JAK-STAT3 target genes are underneath (Chen et al., 2015). Data show the mean ± SD of three biological replicates and were compared with the 0 h time point using an ANOVA test with Dunnett's multiple comparisons test (ns, p > 0.05; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

(E) Prolonged JAK inhibition (dashed lines) strongly impairs the proliferation of naive hPSC (left) with a modest effect on primed hPSC (right) compared with vehicle controls (solid line). Data are shown for two independent experiments.

(F) Flow cytometry plots of live, human cells at day 12 of primed to naive hPSC reprogramming using Chemical Resetting conditions in the absence (Ctrl) and presence (JAKi) of JAK inhibitor. The JAK inhibitor was applied between days 5 and 10 at a final concentration of 1 μM. CD75 and SUSD2 are naive-specific markers and CD24 is a primed-specific marker. Quadrants containing reprogrammed naive cells are boxed in blue. Established naive and primed hPSC are shown as controls.

(G) Phase contrast images of cells at days 1 and 12 during primed to naive hPSC reprogramming in the absence and presence of JAK inhibitor. Scale bar, 100 μm.

Results in (F) and (G) are representative of three biological replicates.