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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression <t>of</t> <t>HOXC9</t> and <t>KDR</t> on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).
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(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression of HOXC9 and KDR on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).

Journal: bioRxiv

Article Title: Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

doi: 10.64898/2026.04.14.718471

Figure Lengend Snippet: (A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression of HOXC9 and KDR on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).

Article Snippet: Following primary antibodies were used; TBXT (Abcam, ab209665, 1:1000), ISL1 (DSHB, 39.4D5, 1:200), HAND1 (R&D Systems, AF3168, 1:100), PITX1 (Novus Bio, NBP1-88644, 1:500), HOXC9 (Abcam, ab50839, 1:200), KDR (R&D Systems, AF357, 1:1000), TP63 (Abcam, ab124762, 1:200).

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Comparison