Fig 1.
Conversion of primed hESCs to the naïve state of pluripotency.
(A) Colony morphology of primed H9 and CHA-hES4 cells and their naïve cells cultured in 2i/LF/A, 2i/L/X/F/P or LCDM media. The scale bar is 100 μm. (B, C) Cell surface expression of primed (SSE-3, SSEA-4, TRA-1-60, TRA-1-81, CD24, and CD90) and naïve markers (CD7, CD77, and CD130) on primed H9 (B) and 2i/L/F/A-naïve hPSCs. (C). Black lines, cell surface marker with the indicated primary antibodies; red area, no primary antibody control.
Fig 2.
Binding profiles of N1-A4 on primed/naïve hPSCs and various cells.
(A, B) Expression of N1-A4 antigen was examined by flow cytometry in primed and naïve hPSCs (A), human embryonic carcinoma cells (NT-2/D1, 2102Ep), hepatocellular carcinoma cells, (Huh7, SNU-387), and MEFs (B). Black lines, cell surface marker with the indicated primary antibody; red area, no primary antibody control.
Fig 3.
(A) Huh7 cell lysates were immunoprecipitated with N1-A4 or goat anti-HSP60 antibodies (α-HSP60), and the immunoprecipitates were detected with α-HSP60 in Western blot analysis. Red arrowhead indicates HSP60. (B) HSP60-Myc/His vector was overexpressed in 293FT cells, and Myc-tagged HSP60 protein was detected with α-Myc. β-actin expression was the loading control. (C, D) HEK293FT cells were transfected with HSP60-Myc/His expression vector. Cell lysates were immunoprecipitated with α-Myc, α-HSP60, and N1-A4 (C), or α-Myc, α-His, and N1-A4 (D). The immunoprecipitates were detected by Western blot analysis with α-Myc. Red arrowhead indicates the HSP60 proteins.
Fig 4.
Expression kinetics of HSP60 during EB differentiation of primed hPSCs.
Primed H9 hPSCs were differentiated by EB formation for 10 days. The expression of OCT4, PAX6, and HSP60 genes was determined by qPCR. qPCR data are presented as relative expression changes considering expression in undifferentiated hPSCs (Day 0) as 1. The graphs represent the mean values of three independent determinations ±SD (ns, not significant; *, p < .05; ***, p < .005).
Fig 5.
Knockdown effects of HSP60 in primed hPSCs.
(A) Cell lysates from HSP60 siRNA-transfected H9 hPSCs were subjected to Western blot analysis with antibodies against HSP60, OCT4, SOX2, and NANOG. Relative protein levels were measured using ImageJ software and normalized to the β-actin. (B) qPCR analysis for the expression of HSP60 and pluripotency genes (OCT4, SOX2, and NANOG) in control (siCtrl) or HSP60 siRNA-transfected H9 hPSCs (siHSP60). Relative mRNA levels were measured by qPCR and were shown after normalization against GAPDH mRNAs. The graphs represent the mean values of at least two independent determinations ±SD (***, p < .005).
Fig 6.
Knockdown effects of HSP60 in naïve hPSCs.
The relative expression of pluripotency genes (OCT4 and NANOG), naïve-state-specific genes (REX1, PRDM14, KLF4, KLF5, and DPPA5), primed-state-specific genes (OTX2), and HSP60 genes was measured in siHSP60-transfected 2i/L/F/A-naïve hPSCs by qPCR. qPCR data are presented as relative expression changes, considering expression in siCtrl-transfected hPSCs as 1. The graphs represent the mean values of two independent determinations ±SD (ns, not significant; *, p < .05; **, p < .01; ***, p < .005).