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. 2021 Aug 31;10:e67259. doi: 10.7554/eLife.67259

Figure 3. Inhibiting TGFβ signalling induces loss of pluripotency in naïve hPSCs.

(a) RT-qPCR expression analysis of pluripotency-associated genes and TGFβ-associated genes in naïve hPSCs (H9 NK2 line) following SB-431542 treatment (t2iLGö + SB). Expression levels are shown as fold changes relative to day 0. (b) Schematic showing the integration of a single-step optimised inducible knock-down targeting construct into the AAVS1 locus of H9 hPSCs, enabling the expression of SMAD2 and SMAD3 short hairpin RNAs (shRNAs) under the control of a tetracycline inducible promoter. ZFN: zinc-finger nucleases; 5’-HAR/3’-HAR: upstream/downstream homology arm; H1-TO: Tetracycline-inducible H1 Pol III promoter carrying one tet operon after the TATA box; CAAG: CMV early enhancer, chicken β-actin and rabbit β-globin hybrid promoter; TetR: Tetracycline-sensitive repressor protein; SA: splice acceptor; Puro, Puromycin resistance; pA, polyadenylation signal. Schematic adapted from Bertero et al., 2016. (c) RT-qPCR analysis of gene expression levels in SMAD2/3 inducible knock-down (iKD) H9 naïve hPSCs following 5 days of tetracycline (tet) treatment. Expression levels are shown for each gene as fold change relative to iKD -tet. Cells were cultured in t2iLGö medium. (d) Phase contrast pictures of H9 NK2 naïve hPSCs after 5, 7, and 10 days of SB treatment in t2iLGö medium. Scale bars: 400 µm. (e) RT-qPCR analysis of trophoblast (HAND1, TP63, MMP2, and SDC1) and pluripotency (POU5F1, NANOG) gene expression levels in naïve hPSCs following long-term (14 days) SB treatment in t2iLGö medium. Expression levels are shown as fold changes relative to day 0 samples, n = two biological replicates. (f) Immunofluorescence microscopy showing the downregulation of NANOG (green) in naïve hPSCs following 3 and 5 days of SB treatment. DAPI signal in blue. White arrowheads indicate colonies displaying heterogeneous expression of NANOG. Scale bars: 50 µm. (g) Immunofluorescence microscopy for OCT4 (red), HAND1 (green), GATA3 (cyan), and DAPI (blue) in naïve hPSCs following 3 and 5 days of SB treatment in t2iLGö medium. Scale bars: 50 µm. (h) Immunofluorescence microscopy for GATA3, HLA-G, SDC1 (magenta), CK19 and CK7 (yellow), and DAPI (blue) in naïve-derived trophoblast stem cells (TS), extravillous trophoblast (EVT), and syncytiotrophoblast (STB). Scale bars: 50 µm. (i) RT-qPCR analysis of gene expression levels in naïve-derived trophoblast stem cells (TS), extravillous trophoblast (EVT) and syncytiotrophoblast (STB) compared to undifferentiated naïve hPSCs. Expression levels are shown for each gene relative to the housekeeping gene RPLP0. RT-qPCR data show the mean ± SD of three biological replicates (unless specified otherwise) and were compared to their relative control using an ANOVA with Tukey's or Šídák's multiple comparisons test (*p ≤ 0.05, **p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001).

Figure 3—source data 1. Numerical data that are represented in Figure 3.

Figure 3.

Figure 3—figure supplement 1. TGFβ signalling inhibition induces loss of pluripotency in different naïve hPSCs.

Figure 3—figure supplement 1.

(a) Heatmap reporting RT-qPCR expression analysis of pluripotency genes and TGFβ-associated genes in naïve cells following the addition of SB to the culture medium (t2iLGö), related to Figure 3a. Data show the mean of three biological replicates as fold changes compared to day 0. (b) Western blot showing TGFβ pathway activation in H9 NK2 naïve cells through the phosphorylation of SMAD2 (pSMAD2) and also total SMAD2/3 in normal conditions (-), after 1 hr and 2 hr of fresh media change (t2iLGö), and following 1 hr and 24 hr of SB treatment (t2iLGö+SB). Alpha-tubulin (α-tub) used as loading control. (c) Phase-contrast images of H9 NK2 naïve cells at day 0 (t2iLGö) and following 5, 7, 8, and 10 days of SB supplementation to the t2iLGö medium. Scale bars: 400 µm. (d) Phase-contrast images of chemically-reset cR-H9 naïve cells after 7 days of SB treatment. (e) Immunofluorescence microscopy of H9 NK2 naïve hPSCs for NANOG (red/green), and DAPI (blue), after 7 days of SB treatment in chemically-reset cR-H9 naïve cells, matching the phase-contrast images in (d). White arrowheads indicate colonies displaying heterogeneous NANOG expression. Scale bars: 50 µm. (f) Immunofluorescence microscopy complementing Figure 3g showing (left) OCT4 (magenta) and HAND1 (yellow) overlap, or (right) OCT4 (magenta) and GATA3 (cyan) overlap after 3 and 5 days of SB treatment. Scale bars: 50 µm. (g) Immunofluorescence microscopy of H9 NK2 naïve hPSCs for OCT4 (red), HAND1 (green), GATA3 (cyan) and DAPI (blue) after 3 (upper) and 5 (lower) days of SB treatment. Scale bars: 50 µm.
Figure 3—figure supplement 1—source data 1. Full uncropped western blot from Figure 3—figure supplement 1b reporting TGFβ pathway activation in H9 NK2 naïve cells through the phosphorylation of SMAD2 (pSMAD2) and also total SMAD2/3 in normal conditions (-), after 1 hr and 2 hr of fresh media change (t2iLGö), and following 1 hr and 24 hr of SB treatment (t2iLGö+SB).
Alpha-tubulin (α-tub) used as loading control. Additional lanes (not shown in the main figure for clarity) report as positive control naïve (N) and primed (P) hPSC treated for 1 hr with Activin. (a) original files of the full raw unedited blots. (b) figures with the uncropped blots with the bands reported in the main figures highlighted in red and labelled accordingly.