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Stem cell conversion to the cardiac lineage requires nucleotide signalling from apoptosing cells

Abstract

Pluripotent stem cells can be driven by manipulation of Wnt signalling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic-mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that initiation of differentiation in induced pluripotent stem cells (iPSCs) and embryonic stem cells triggers widespread apoptosis, followed by a synchronous epithelial–mesenchymal transition (EMT). Apoptosis is caused by the absence of bFGF in the differentiation medium. EMT requires induction of the transcription factors SNAI1 and SNAI2 downstream of MESP1 expression, and double knockout of SNAI1 and SNAI2 or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis, either chemically or by ablation of pro-apoptotic genes, also completely prevents EMT, suppressing even the earliest events in mesoderm conversion, including T/BRA, TBX6 and MESP1 induction. Conditioned medium from WNT-activated wild-type iPSCs overcomes the block to EMT by cells incapable of apoptosis, suggesting involvement of soluble factors from apoptotic cells in mesoderm conversion. Knockout of the PANX1 channel blocked EMT, whereas treatment with a purinergic P2-receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signalling. ATP and/or UTP was sufficient to induce a partial EMT in apoptosis-incapable cells treated with WNT activator. Notably, knockout of the ATP/UTP-specific P2Y2 receptor blocked EMT and mesoderm induction. We conclude that in addition to acting as chemo-attractants for clearance of apoptotic cells, nucleotides can function as essential paracrine signals that, with WNT signalling, create a logical AND gate for mesoderm specification.

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Fig. 1: Mesoderm induction triggers apoptosis followed by EMT.
Fig. 2: bFGF withdrawal induces apoptosis but not cell conversion.
Fig. 3: MESP1-induced Snail/Slug expression is required for EMT and cardiogenesis.
Fig. 4: Apoptosis is required for WNT-mediated EMT.
Fig. 5: Apoptosis licenses early mesoderm commitment.
Fig. 6: Soluble factors released through Pannexin-1 channels license surviving iPSCs to respond to WNT activation.
Fig. 7: ATP and/or UTP license mesoderm specification.
Fig. 8: P2Y2 receptors are required for mesoderm specification, EMT and cardiogenesis.

Data availability

The previously published RNA-sequencing data that were re-analysed in Extended Data Fig. 8a are available from the Allen Institute for Cell Science (https://www.allencell.org/genomics.html). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank P. Joshi (Gama laboratory) for establishing the BAX/BAK DKO iPSCs and M. Rasmussen (Gama laboratory) for the providing the SIM pictures of iPSC-derived cardiomyocytes. We thank the members of the Macara laboratory for discussion. This work was supported by grant nos GM070902 from NIGMS and CA197571 from the NCI (both to I.G.M.) as well as NIH grant no. 1R35GM128915-01 (to V.G.).

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization: L.F., V.G. and I.G.M. Methodology: L.F. and I.G.M. L.F. performed experiments and analysed data. V.G. provided resources. L.F. prepared the figures. I.G.M. and L.F. wrote and edited the manuscript. I.G.M. supervised the work.

Corresponding author

Correspondence to Ian G. Macara.

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The authors declare no competing interests.

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Nature Cell Biology thanks Thomas Zwaka, and the other, anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1. Pluripotent stem cells undergo EMT during conversion to cardiac mesoderm.

A) Representative immunoblot of WT hiPSC for pluripotency markers (Nanog, Oct4, Sox2), tight junctions marker (ZO-1), Crumb complex (PALS1), PAR complex (PAR-3, PKC-ζ), Scribble complex (LLGL2, Scribble) and α-Tubulin. Molecular weights (M.W.) are indicated in kDa. B) Relative gene expression of NANOG, TBXT, NKX2.5, ANP, ISL1 during differentiation. ΔΔCt values were normalized to the mTeSR1 condition (prior to cell differentiation). Independent biological repeats are colour-coded (N=3 independent experiments). Mean ± s.e.m. C) Relative gene expression of TBXT, EOMES, TBX6, MESP1, MESP2 during CHIR induction. ΔΔCt values were normalized to the mTseSR1 condition (prior to cell differentiation). Independent biological repeats are colour-coded (N=3 independent experiments). Mean ± s.e.m. D) Representative immunoblot of hiPSCs and hiPSC-derived cardiomyocytes obtained 12 days post differentiation (Top pathway described in Fig. 1B). Expression of EMT markers (Slug and E-Cadherin) were analysed. Molecular weights (M.W.) are indicated in kDa. (N=2 independent experiments). E) Relative gene expression of VIM was analysed by qRT-PCR during cell conversion. ΔΔCt values were normalized to the mTseSR1 condition. Independent biological repeats are colour-coded (N=3 independent experiments). Mean ± s.e.m. F) Representative Max IPs images of wildtype hiPSCs fixed at the indicated times along the differentiation protocol and stained for F-actin (Phalloidin) and nuclei (Hoechst). Scale bar = 50 μm. Magnified area of the F-actin channel (yellow dotted square) is shown. Scale bar = 10 μm. G) Representative Immunoblot comparing expression of EMT markers (E-Cadherin, Snail, Slug) and cardiac marker (HAND1) between the two protocols shown in Fig. 1B. H-I) Immunoblot of PARP cleavage in hESC H9 during CHIR treatment. Molecular weights (M.W.) are indicated in kDa (H). PARP cleavage was quantified by densitometry across 2 independent biological repeats (colour-coded). Tukey’s multiple comparison was applied (I). J) Snail and Slug expression was analysed in hESC H9 following CHIR induction. Molecular weights (M.W.) are indicated in kDa. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2. bFGF withdrawal induces apoptosis randomly across the cell population.

A) Representative immunoblot of PARP cleavage of hiPSCs treated with RPMI/B27(-Ins) supplemented or not with CHIR and collected at the indicated time. Molecular weights (M.W.) are indicated in kDa. B) Clark-Evans analysis was used to measure randomness of cell death events after 24hrs CHIR treatment (See Methods). Cleaved Caspase-3 staining distribution was analysed and compared to a theorical random distribution (R=1). R values closer to 0 have a tendency towards clustering while R values closer to 2 represent a tendency towards regularity. Two-tailed Wilcoxon Signed Rank Test (N=3 independent experiments). C-D) Representative Max IPs pictures of hiPSC treated with CHIR and stained for nuclei and c-Myc. Scale bar = 50 μm (C). c-Myc expression was analysed and presented as violin plots (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=5 random fields of view obtained from 2 independent experiments) (D). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3. Density-dependent pathways do significantly promote EMT.

A-B) Representative Max IPs pictures of hiPSC treated with CHIR and BrdU 1h prior to fixation. Cells were stained for nuclei and BrdU to assess cell proliferation. Scale bar = 50 μm (A). BrdU incorporation was analysed and presented as violin plots (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=10 random fields of view obtained from 3 independent experiments). Dunn’s multiple comparisons test was applied. (B). C-D) Representative Max IPs pictures of hiPSC treated with CHIR and stained for nuclei, ZO-1 and YAP. Scale bar = 50 μm (C). Nuclear-to-cytosolic ratio of YAP was analysed and presented as violin plots (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=15 random fields of view obtained from 3 independent experiments). Dunn’s multiple comparisons test was applied (D). Source numerical data are available in source data.

Source data

Extended Data Fig. 4. Apoptosis is required for EMT and cardiac differentiation.

A) Representative Max IPs images of hESC H9 co-treated with CHIR + DMSO (left) or CHIR + 10 μM Q-VD-OPH (right) stained for ZO-1 (green), cleaved caspase 3 (yellow) and nuclei (blue). Scale bar = 50 μm. Magnified area (yellow dotted square) is shown as a merge. Scale bar = 10 μm. B-C) Immunoblot analysis of hESC H9 co-treated with CHIR + DMSO or 10 μM Q-VD-OPH. Molecular weights (M.W.) are indicated in kDa (B). Normalized expression of Snail and Slug was quantified by densitometry across 3 independent biological replicates (colour-coded). Mean ± s.e.m. (C). D) Immunoblot of CASP3 and CASP9 KO cell lines (non clonal). Knockout validation was performed by probing for Caspase 3 and Caspase 9 expression, as well as Nanog as a stem cell marker. Molecular weights (M.W.) are indicated in kDa. E) Immunoblot of control Non Targeted (NT2) and CASP3 and CASP9 KO cell lines, analysed for Snail and Slug expression upon CHIR induction. Molecular weights (M.W.) are indicated in kDa. F) Representative Max IPs images of Non targeted (NT2) and CASP3 KO hiPSCs, induced 72hrs with CHIR and stained for ZO-1 (green), Slug (magenta) and nuclei (blue). Scale bar = 50 μm. Magnified area (yellow dotted square) is shown as a merge. Scale bar = 10 μm. G-H) Violin plots representing numbers of nuclei over time for WT hiPSCs co-treated with CHIR ± Q-VD-OPH (G) or BAX/BAK DKO hiPSCs treated with CHIR. (Median: plain red line – Quartiles: black dotted lines). Three independent biological repeats are colour-coded (Number of fields of view: DMSO 0hrs N=14, DMSO 24hrs N=39, DMSO 48hrs N=19, DMSO 72hrs N=18, Q-VD 0hrs N=14, Q-VD 24hrs N=39, Q-VD 48hrs N=21, Q-VD 72hrs N=15) (Number of fields of view Ctr 0hrs N=14, Ctr 24hrs N=34, Ctr 48hrs N=20, Ctr 72hrs N=14, DKO 0hrs N=14, DKO 24hrs N=33, DKO 48hrs N=20, DKO 72hrs N=14). I) SIM images of isogenic control and BAX/BAK DKO hiPSC-derived cardiomyocytes, plated at low density and stained for alpha-actinin. Scale bar = 5 μm. Magnified area (yellow dotted square) is shown. Scale bar = 1 μm. J) Immunoblot analysis of hiPSC treated with CHIR and IWP-2 co-treated or not with 10 μM Q-VD-OPH and probed for cleaved Caspase-3 and cardiac marker HAND1. Cell lysates were collected as indicated on the timeline (red arrows). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 5. Apoptosis is required early in selection of mesoderm lineage.

A-B) Representative Max IPs images of hiPSCs stained for T/Bra (top) and EOMES (bottom) and nuclei, before (0hrs) and after CHIR treatment ± Q-VD-OPH. Scale bar = 50 μm. (A). Violin plots summarize quantification of the percentage of T/Bra-positive (top) and EOMES-positive (bottom) nuclei after CHIR treatment (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=19 and 21 random fields of view for EOMES staining in DMSO and Q-VD-treated cells respectively, obtained from 2 independent experiments and N=39 random fields of view for T/Bra staining, obtained from 3 independent experiments). Two-tailed Mann–Whitney test was applied. C) Relative gene expression of EOMES, TBXT, MESP1, TBX6 obtained from WT hiPSCs, induced or not with CHIR and DMSO or CHIR and Q-VD-OPH for 46hrs and analysed by qRT-PCR. ΔΔCt values were normalized to uninduced cells. Independent biological repeats are colour-coded (N=3 independent experiments for EOMES and N=4 independent experiments for the remaining data set). Error bar = Mean ± s.e.m. Two-tailed paired t-test was applied to compare 0hrs vs 46hrs and unpaired t-test was applied to compare DMSO vs Q-VD. D-G) Representative Max IPs images of control NT2, CASP3 and CASP9 KO hiPSCs stained for T/Bra (D), EOMES (F) and nuclei after CHIR treatment. Scale bar = 50 μm. Percentage of T/Bra-positive (E) and EOMES-positive (G) nuclei after CHIR treatment are shown as violin plots (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=20 random fields of view obtained from 3 independent experiments). Kurskal-Wallis test was applied. H) Timeline for the Q-VD-OPH treatment and lysate collection/fixation time points. hiPSCs were treated with CHIR only for 24hrs, before adding CHIR +/− 10 μM Q-VD-OPH for another 28hrs and 48hrs with CHIR (52hrs and 72hrs time point respectively). I) Immunoblot analysis of hiPSCs treated as presented in (H) and probed for EMT markers (Snail and Slug) and cell death markers (PARP and cleaved Caspase-3). Molecular weights (M.W.) are indicated in kDa. J) Representative Max IPs images of hiPSCs treated as presented in (H) and stained for ZO-1 (green) and Slug (magenta). Scale bar = 50 μm. Magnified area (yellow dotted square) is shown as a merge (bottom row). Scale bar = 10 μm. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 6. Small molecule inhibitor of Pannexin and gap junctions blocks mesoderm specification and cardiac differentiation.

A-C) Representative Max IPs pictures of hiPSC co-treated with CHIR + Vehicle (water) or CHIR 100 μM Carbenoxolone for 72hrs and stained for nuclei (blue), ZO-1 (green) and Slug (Magenta). Scale bar = 50 μm. Magnified area (yellow dotted square) is shown as a merge. Scale bar = 50 μm (A). Percentage of area with intact tight junctions and percentage of Slug-positive nuclei is presented as violin plots in (B) and (C) respectively. (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=8 random fields of view obtained from 2 independent experiments). Two-tailed unpaired t-test was applied (B-C). D) Immunoblot analysis of Snail and Slug expression level during CHIR treatment in the presence or absence of Carbenoxolone. Molecular weights (M.W.) are indicated in kDa. (N=2 independent repeats). E) Complete cardiomyocyte differentiation protocol timeline. 100 μM Carbenoxolone or Vehicle (water) was supplemented to the media during the CHIR step for 48hrs, followed by the usual steps and maintained in RPMI/B27(+Ins) media. H) hiPSC were treated as presented in E and analysed for expression of cardiac markers (Cardiac Troponin T and Nkx2.5) 13 days post differentiation initiation. Molecular weights (M.W.) are indicated in kDa. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 7. Apyrase treatment partially blocks EMT.

A) Timeline of apyrase-treated conditioned media (CM) experiment. WT hiPSC were initially treated with CHIR + Q-VD-OPH. At the same time, WT hiPSCs were treated with CHIR only. 24hrs later, Q-VD-OPH was washed out and condition media from the CHIR-only treatment was added onto the Q-VD-OPH pre-treated cells, in the presence or absence of 2U/mL of Apyrase. B) Representative Max IPs pictures of WT cells fixed after adding apyrase-treated CM as presented in (A). Cells were stained for ZO-1, Slug and nuclei. Scale bar = 50 μm. Magnified area (yellow dotted square) is shown for the ZO-1 channel. Scale bar = 10 μm.

Extended Data Fig. 8. P2Y2 receptor signalling is required for mesoderm specification.

A) Gene expression for different members of the P2Y receptor family was obtained from RNAseq data realized by the Allen Institute. Data were obtained using the GM25256 hiPSC cell line, sequenced at different passage (colour-coded). B-E) Representative Max IPs images of control (NT2) and P2RY2 KO hiPSCs treated with CHIR for 52hrs and stained for nuclei and T/Bra (B) or EOMES (D). Scale bar = 50 μm. (B-D). Percentage of T/Bra (C) and EOMES-positive (E) nuclei were quantified and displayed as violin plots (Median: plain red line – Quartiles: black dotted lines). Independent biological repeats are colour-coded (N=15 random fields of view obtained from 3 independent experiments). Dunn’s multiple comparisons test comparison test was applied in (C) and (E). Source numerical data are available in source data.

Source data

Supplementary information

Supplementary Information

Gating strategy corresponding to the Annexin V–APC assay presented in Fig. 1h. Values corresponding to quadrant Q4 (PIAnnexin V–APC+) are reported on each graph.

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Supplementary Table

Supplementary Tables 1–4.

Supplementary Video 1

Phase contrast time lapse of iPSC-derived cardiomyocytes obtained using the GiWi differentiation protocol. Spontaneous beating was observed 12 d after protocol initiation and immature cardiomyocytes were maintained in RPMI/B27(+Ins) medium.

Supplementary Video 2

Time-lapse imaging of mEGFP–TJP1 knock-in hiPSCs, starting 40 h after CHIR treatment. Scale bar, 50 μm. MIPs are shown.

Supplementary Video 3

Bright-field time-lapse imaging of control (NT2) and MESP1-KO (gRNA 1 and 2) human iPSC-derived cardiomyocytes. Videos were recorded 12 days after GiWi-protocol initiation using an EVOS FL microscope (×10 objective). Scale bar, 400 μm.

Supplementary Video 4

Bright-field time-lapse imaging of control (Ctr3) and BAX/BAK-DKO iPSC-derived cardiomyocytes. Videos were recorded 11 d after GiWi-protocol initiation using an EVOS FL microscope (×4 objective). Scale bar, 1,000 μm.

Supplementary Video 5

Bright-field time-lapse imaging of control (NT2) and PANX1-KO iPSC-derived cardiomyocytes. Videos were recorded 12 d after GiWi-protocol initiation using an EVOS FL microscope (×10 objective). Scale bar, 400 μm.

Supplementary Video 6

Bright-field time-lapse imaging of control (vehicle)- and carbenoxolone-treated iPSC-derived cardiomyocytes. Treatment was applied during the first 48 h of differentiation, as depicted in Supplementary Fig. 6e. Videos were recorded 13 d after GiWi-protocol initiation using an EVOS FL microscope (×10 objective). Scale bar, 400 μm.

Supplementary Video 7

Bright-field time-lapse imaging of control (vehicle)- and suramin-treated iPSC-derived cardiomyocytes. Treatment was applied during the first 48 h of differentiation, as depicted in Fig. 8j. Videos were recorded 12 d after GiWi-protocol initiation using an EVOS FL microscope (×10 objective). Scale bar, 400 μm.

Supplementary Video 8

Bright-field time-lapse imaging of control (NT2) and P2RY2-KO iPSC-derived cardiomyocytes. Videos were recorded 12 d after GiWi-protocol initiation using an EVOS FL microscope (×10 objective). Scale bar, 400 μm.

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Fort, L., Gama, V. & Macara, I.G. Stem cell conversion to the cardiac lineage requires nucleotide signalling from apoptosing cells. Nat Cell Biol 24, 434–447 (2022). https://doi.org/10.1038/s41556-022-00888-x

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