Journal: bioRxiv
Article Title: Human pluripotent stem cell-derived atrioventricular node-like pacemaker cells exhibit biological conduction bridge properties in vitro and in vivo
doi: 10.1101/2025.09.04.674322
Figure Lengend Snippet: A. Representative flow cytometric analysis and corresponding summary of CD235a and PDGFRα expression in day 4 mesoderm following induction with the specified concentrations of BMP4 (B) and Activin A (A) [ng/ml] in the HES2 cell line (n = 14). B. Representative flow cytometric analysis and corresponding summary of CD235a and PDGFRα expression in day 4 mesoderm following induction with the specified concentrations of BMP4 (B) and Activin A (A) [ng/ml] in the ESI017 ASAP1 cell line (n = 15-16). C. Representative flow cytometric analysis and corresponding summary of cTNT and MLC2V expression in day 20 populations generated with the indicated amounts of BMP4 (B) and Activin A (A) [ng/ml] during mesoderm induction (days 1-3) in the HES2 cell line (n = 14). D. Representative flow cytometric analysis and corresponding summary of cTNT and MLC2V expression in day 20 populations generated with the indicated amounts of BMP4 (B) and Activin A (A) [ng/ml] during mesoderm induction (days 1-3) in the ESI017 ASAP1 cell line (n = 15-16). E. Representative flow cytometric analysis for the expression of cTNT in day 20 cultures treated with either 12 μM of CHIR or 200 ng/mL of BMP2 at the indicated timepoints in the HES2 cell line. F. Bar graph summarizing the proportion of cTNT + cells shown in F (n = 14). G. Representative flow cytometric analysis for the expression of cTNT in day 20 cultures treated with either 12 μM of CHIR or 200 ng/mL of BMP2 at the indicated timepoints in the ESI-017 ASAP1 cell line. H. Bar graph summarizing the the proportion of cTNT + cells shown in G (n = 9). I. Heatmap showing differences in RT-qPCR gene expression levels across HES3, HES2 and ASAP1 AVNLPCs treated with either 12 μM of CHIR (CHIR AVNLPCs) or 200 ng/mL of BMP2 (BMP2 AVNLPCs). VLCMs differentiated from each cell line were included as an expression reference. All populations were analyzed at day 20 of differentiation. Values represent log2 of expression levels relative to the housekeeping gene TBP. J. Immunofluorescent staining of MSX2 in BMP2 AVNLPCs and VLCMs from the HES2 and ESI-017 ASAP1 cell lines. Cells were counterstained with cTNT to visualize all cardiomyocytes and DAPI to visualize all cells. Scale bars, 100 μm. K. Quantification of the percentage of MSX2 + cells determined by immunofluorescence staining in BMP2 AVNLPCs and VLCMs from the HES2 and ESI-017 ASAP1 cell lines (n = 4-5 images per monolayer, taken from 3 independent differentiations). Error bars represent SEM. Statistical tests: One-way ANOVA, Bonferroni’s post hoc test (F, H); unpaired two-sided t-test (A-D, K): *P < 0.05, **P < 0.01, ***P < 0.001.
Article Snippet: The HES2 cell line (karyotype: 46, XX) was purchased from WiCell.
Techniques: Expressing, Generated, Quantitative RT-PCR, Gene Expression, Staining, Immunofluorescence
WiCell Research Institute Inc is a verified supplier 
WiCell Research Institute Inc manufactures this product 
![A. Representative flow cytometric analysis and corresponding summary of CD235a and PDGFRα expression in day 4 mesoderm following induction with the specified concentrations of BMP4 (B) and Activin A (A) [ng/ml] in the <t>HES2</t> cell line (n = 14). B. Representative flow cytometric analysis and corresponding summary of CD235a and PDGFRα expression in day 4 mesoderm following induction with the specified concentrations of BMP4 (B) and Activin A (A) [ng/ml] in the ESI017 ASAP1 cell line (n = 15-16). C. Representative flow cytometric analysis and corresponding summary of cTNT and MLC2V expression in day 20 populations generated with the indicated amounts of BMP4 (B) and Activin A (A) [ng/ml] during mesoderm induction (days 1-3) in the HES2 cell line (n = 14). D. Representative flow cytometric analysis and corresponding summary of cTNT and MLC2V expression in day 20 populations generated with the indicated amounts of BMP4 (B) and Activin A (A) [ng/ml] during mesoderm induction (days 1-3) in the ESI017 ASAP1 cell line (n = 15-16). E. Representative flow cytometric analysis for the expression of cTNT in day 20 cultures treated with either 12 μM of CHIR or 200 ng/mL of BMP2 at the indicated timepoints in the HES2 cell line. F. Bar graph summarizing the proportion of cTNT + cells shown in F (n = 14). G. Representative flow cytometric analysis for the expression of cTNT in day 20 cultures treated with either 12 μM of CHIR or 200 ng/mL of BMP2 at the indicated timepoints in the ESI-017 ASAP1 cell line. H. Bar graph summarizing the the proportion of cTNT + cells shown in G (n = 9). I. Heatmap showing differences in RT-qPCR gene expression levels across HES3, HES2 and ASAP1 AVNLPCs treated with either 12 μM of CHIR (CHIR AVNLPCs) or 200 ng/mL of BMP2 (BMP2 AVNLPCs). VLCMs differentiated from each cell line were included as an expression reference. All populations were analyzed at day 20 of differentiation. Values represent log2 of expression levels relative to the housekeeping gene TBP. J. Immunofluorescent staining of MSX2 in BMP2 AVNLPCs and VLCMs from the HES2 and ESI-017 ASAP1 cell lines. Cells were counterstained with cTNT to visualize all cardiomyocytes and DAPI to visualize all cells. Scale bars, 100 μm. K. Quantification of the percentage of MSX2 + cells determined by immunofluorescence staining in BMP2 AVNLPCs and VLCMs from the HES2 and ESI-017 ASAP1 cell lines (n = 4-5 images per monolayer, taken from 3 independent differentiations). Error bars represent SEM. Statistical tests: One-way ANOVA, Bonferroni’s post hoc test (F, H); unpaired two-sided t-test (A-D, K): *P < 0.05, **P < 0.01, ***P < 0.001.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_22/10__1101_slash_2025__09__04__674322/10__1101_slash_2025__09__04__674322___F3.large.jpg)
