Supplementary Figure 3: BMP signaling promotes SANLPC development. | Nature Biotechnology

Supplementary Figure 3: BMP signaling promotes SANLPC development.

From: Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker

Supplementary Figure 3

(a) The total number of cTNT+ cells and the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells in day 20 populations generated from mesoderm induced with the indicated amounts of BMP and ACTA between days 1-3 of differentiation (n = 3). (b) Flow cytometric analyses showing the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells in day 20 populations generated with and without (Control) the addition of the TGFβ/Activin/Nodal inhibitor SB-431542 (SB) (5.4 μM) between days 3-5 from mesoderm that was induced with 3 ng/ml BMP4 and 2 ng/ml ACTA (n = 4). (c-e) Flow cytometric analyses of the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells in day 20 populations that were specified by addition of the indicated amounts of dorsomorphin or BMP4 together with SB (5.4 μM) between days 3-5 from mesoderm that was induced with either 3 ng/ml BMP4, 2 ng/ml ACTA (3B/2A) (n = 4) (c) or 5 ng/ml BMP4, 4 ng/ml ACTA (5B/4A) (n = 3) (d) or 10 ng/ml BMP4, 6 ng/ml ACTA (10B/6A) (n = 4) (e). t-test: *P < 0.05, **P < 0.01 vs Nkx2-5-cTNT+ cells at endogenous (E) BMP4 levels. (f) Total number of cells in day 20 populations specified from mesoderm by addition of the indicated amounts of dorsomorphin or BMP4 together with SB (5.4 μM) between days 3-5 (n = 4). (g) qRT-PCR analysis of TBX18 expression in populations at indicated time points specified from mesoderm with no additional treatment (control) or by the addition of 2.5 ng/ml BMP4 + 5.4 μM SB (BMP) (days 3-5). t-test: **P < 0.01 vs indicated sample (n = 4). (h) Quantification of TBX18+ cells in immunostained day 6 populations specified from mesoderm with no additional treatment (control) or by the addition of 2.5 ng/ml BMP4 + 5.4 μM SB (BMP) (days 3-5). t-test: **P < 0.01 vs control (n = 11 images from N = 3 cell culture replicates). (i) Photomicrograph showing immunostaining of TBX18 in day 6 populations specified from mesoderm with no additional treatment (control) or by the addition of 2.5 ng/ml BMP4 + 5.4 μM SB (BMP) (days 3-5). DAPI was used to visualize cell nuclei. Scale bars represent 100 μm. All error bars represent s.e.m. d, day; Dorso, dorsomorphin; E, endogenous

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