Additional file 1 of Chemically defined and small molecules-based generation of sinoatrial node-like cells
Creators: name: Hou, Xiaojie
name: Ma, Shuhong
name: Fan, Wei
name: Li, Fang
name: Xu, Miaomiao
name: Yang, Chao
name: Liu, Feng
name: Yan, Ying
name: Wan, Juyi
name: Lan, Feng
name: Liao, Bin
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Fig. S1. Characterization of the hPSC-derived cardiomyocytes. (A) Morphological contrast image of different hPSC lines and hPSC-derived cardiomyocytes during chemically defined differentiation. (B) Representative flow cytometric analyses of the proportion of cTNT+ cells derived from different hPSC lines at day 24. (C) The hPSC-induced cells expressed α-actinin and cTNT indicated by immunofluorescent staining at day 24 of differentiation. Bar=50 μm. Fig. S2. Gene expression during chemically defined cardiac differentiation. Real-time PCR for markers of mesoderm (TBXT and MIXL1), cardiac mesoderm (MESP1), committed cardiac progenitors (NKX2-5, ISL1, and TBX5), and cardiomyocytes (MYH6, MYH7, and TNNT2). Values represent expression levels relative to the housekeeping gene GAPDH (n = 4). Fig. S3. Expression of SHOX2 and cTNT in SANLPCs. (A, B) Representative flow cytometric analyses of the proportion of SHOX2+/cTNT+ cells for control and PBC group at day 24. (n = 3). Mean ± SEM. *p &lt; 0.05 by Student’s t test. (C, D) Immunofluorescent staining for SHOX2 and cTNT in PBC group, Bar=250 μm, (n = 3). Mean ± SEM. *p &lt; 0.05 by Student’s t test. (E, F) Representative flow cytometric analyses of the proportion of SHOX2+/cTNT+ cells following metabolic selection or not at day 32 of differentiation. (n = 3). Mean ± SEM. *p &lt; 0.05 by Student’s t test. Fig. S4. Generation of SANLPCs from different hPSC lines. (A) Relative expression of SAN gene for PBC group and control group in H9 and UiPS cell lines. (B-E) Representative flow cytometric analyses of the proportion of NKX2.5-/cTNT+ cells for control and PBC group at day 24 of differentiation in H9 and UiPS cell lines. Bar graph indicates average proportion of NKX2-5−/cTNT+ cells for control and PBC group from independent experiments (n = 3). Mean ± SEM. *p &lt; 0.05 by Student’s t test. Fig. S5. The side-by-side comparisons between already available protocols (CHIR-protocol and BPM-protocol) and our current protocol. (A) Heatmap shows expression changes of genes related to SAN cells at day 24 via qPCR. (B-C) Representative flow cytometric analyses of the proportion of NKX2-5−/cTNT+ cells from three different protocols at day 32. **P ≤ 0.01 by Student’s t test (n = 3). All error bars represent the SD of three or four independent experiments. Fig. S6. Electrophysiological characterization of SANLCs generated by three different protocols. (A) Representative action potential recording using whole-cell patch from CHIR-protocol, BPM-protocol, and our current protocol. (B) Patch clamp recordings of induced cells from CHIR-protocol (15 cells), BPM-protocol (20 cells), and our current protocol (20 cells), demonstrating MDP, peak voltage, APA, APD90, APD50, and dV/dtmax (maximal rate of depolarization). (C) Bar graph indicates proportions of cardiomyocyte subtypes at day 32. MDP, maximum diastolic potential; APA, action potential amplitude; APD, action potential duration at different levels of repolarization (90% and 50%).