Fig. 6: Prolonged and combined activation of WNT and BMP signaling pathways during the first stages of hPSC differentiation induced posterior SHF/splanchnic mesoderm specification potentially through RA signaling activation.

a Volcano Plot highlighting the differentially expressed genes in D3WB vs D3 condition. b Gene expression profile of early mesoderm markers T and MESP1, paraxial mesoderm markers TBX6, CDX2, and MSGN1 and the RA synthesizing enzyme ALDH1A2 in control and WB conditions. Values are normalized to GAPDH and D0 of differentiation. Data are represented as mean ± SEM of n = 3 (TBXT, CDX2, ALDH1A2 Control), n = 4 (TBX6, MSGN1, ALDH1A2 D3WB), n = 5 (MESP1 WB, ALDH1A2 D5WB), and n = 6 (MESP1 Control) independent experiments. Exact p-values: TBXT p = 0.0034; CDX2 p = 0.0028; TBX6 p = 0.0305; MSGN1 p < 0.0001; MESP1 p = 0.0090; ALDH1A2 D3 control vs WB p = 0.0427, ALDH1A2 D5 control vs WB p = 0.0001. c Volcano Plot highlighting the differentially expressed genes in D5WB vs D5 condition. d Gene expression profile of TBX5, GATA4, HAND2, MAB21L2, and FOXF1 at D5 and D5WB conditions. Data are represented as mean ± SEM of n = 4 (TBX5 D5WB, GATA4 D5WB, MAB21L2 D5WB), n = 3 (GATA4 D5, HAND2 D5, FOXF1 D5, MAB21L2 D5), n = 5 (TBX5 D5), n = 6 (HAND2 D5WB, FOXF1 D5WB) independent experiments. Exact p-values: TBX5 p = 0.0290; GATA4 p = 0.0121; HAND2 p = 0.0275; MAB21L2 p = 0.0242; FOXF1 p = 0.0008. e Top gene ontology (GO) terms for biological processes identified (FDR < 0.05) of differentially down-regulated genes (Log₂ FC < −1 and adjusted p-value < 0.05) for D5WB vs D5. f Cluster analysis highlighting GO term “Positive regulation of secondary heart field cardioblast proliferation”. In orange are highlighted the genes that were significantly down-regulated in D5WB vs D5 analysis. g Gene expression profile of NKX2.5, TBX1, and TBX20 at D5 and D5WB conditions. Data are represented as mean ± SEM of n = 4 (NKX2.5 D5, TBX1 D5WB, TBX20 D5WB), n = 3 (TBX1 D5, TBX20 D5) and n = 5 (NKX2.5 D5WB) independent experiments. Exact p-values: NKX2.5 p = 0.0005; TBX1 p = 0.0021; TBX20 p = 0.0070. h Cluster analysis highlighting GO terms: “Endodermal cell fate specification”. In orange are highlighted the genes that were significantly down-regulated in D5WB vs D5. i Gene expression profile of SOX17, EOMES, and HHEX at D3/5 and D3/5WB conditions. Data are represented as mean ± SEM of n = 3 (SOX17 D3, EOMES D5/D3WB/D5WB, HHEX), n = 4 (EOMES D3) and n = 6 (SOX17 D3WB) independent experiments. Exact p-values: SOX17 p = 0.0006; EOMES D3 Control vs D3WB p = 0.0335, D5 Control vs D5WB p < 0.0001; HHEX p = 0.0073. j Schematic representation highlighting the main mechanistic outcomes retrieved from the bulk-RNAseq data. k Scheme illustrating the process of CM specification and PE induction from D5WB progenitor cell population. l Percentage of cTnT⁺ cells after 11 days of CM differentiation protocol using D5 and D5WB progenitor cell populations. Data are represented as mean ± SEM of n = 5 (D11) and n = 6 (D11WB) independent experiments. Exact p-value = 0.0006. m Percentage of WT1⁺ cells after 11 days of PE induction from D5 and D5WB progenitor cell populations. Data are represented as mean ± SEM of n = 10 independent experiments. Exact p-value = 0.0087. n Representative IF staining of D11 organoid sections obtained using D5WB progenitor cells. Scale bars, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.