Fig 2: Sfrp2 induced cardiac differentiation. (a) Schematic of cardiac differentiation from human iPSc. (b) Cardiac differentiation was determined by flow cytometry of the cardiomyocyte marker cardiac troponin-T (cTnT). Cells were analyzed on day 14 of the protocol shown in (a). N = 5.

Fig 3: Sfrp2 promotes cardiomyocyte differentiation via of Wnt3a inhibition. (a) On day 3 of the differentiation protocol, cells were incubated with either WntC59 (2 μM) or Sfrp2 (1 nM) for 2 days. After two days, cell extracts were analyzed for total-β-catenin by immunoblotting. Total-β-catenin levels were normalized to the housekeeping protein GAPDH and are shown as a fold change compared to the control. Control protein extracts were isolated from cells prior to the addition of WntC59/Sfrp2. Representative immunoblots are shown. N = 3. Comparisons are made to control cells: ***P < 0.001. An image showing the immunoblot edge is shown in Supplementary Fig. 1. Note, unlike the images provided here, the image in Supplementary Fig. 1 is non-linear data. As such, it is unsuitable for quantification purposes. (b & c) On day 3 of the differentiation protocol, cells were incubated with a human Wnt3a neutralizing antibody (nAb) for 48 h. Cardiomyocyte differentiation was then assessed (day 14) by flow cytometry for cTnT. Representative flow traces are shown in (b) with quantification of the number of cardiomyocytes provided in (c). N = 4. Comparisons made to control cells (antibody vehicle): **P < 0.01. (d) On day 3 of the differentiation protocol, cells were incubated with WntC59 (2 μm) and the indicated doses of human Wnt3a protein for 48 h. Cardiomyocyte differentiation was assessed (day 14) by flow cytometry for cTnT and the fold change determined by normalizing to the control group (WntC59 treatment and Wnt3a protein vehicle). N = 4. Comparisons are made to the control group: ***P < 0.001. (e) On day 3 of the differentiation protocol, cells were incubated with human Wnt3a protein (100 ng/ml) and either vehicle or or Sfrp2 (1 nM) for 48 h. Cardiomyocyte differentiation was assessed (day 14) by flow cytometry for cTnT and the number of cardiomyocytes are expressed as a percentage of the total cell population. N = 3. Comparisons are made to the control group: **P < 0.01.

Fig 4: Sfrp2 improves iPSc-derived cardiomyocyte maturation. iPSc were differentiated into cardiomyocytes via the standard broad-spectrum Wnt inhibitor (WntC59, XAV939) method or via the Sfrp2 method. Cells were analyzed on day 14 of differentiation protocol. (a) cells were fixed and stained with an antibody targeting α-Actinin (Actn2). Left hand side: Representative images shown (scale bar 20 microns). Right hand side: Quantification of two parameters of maturation: circularity and sarcomere length. N > 30 for each group. Comparisons are made to the WntC59 group: ***P < 0.001, *P < 0.05, ns-not significant. (b) Representative image of a Sfrp2-derived cardiomyocyte after 40 days in culture. Scale bar 20 microns. N = 3. (c) Beating frequency was determined on day-14 of the differentiation protocol by measuring the number of contractions over a 20 s period. For each experiment, 3 wells were analyzed. Representative experiment shown. *P < 0.05. (d) Fourteen days after the start of differentiation, patch-clamp was performed. Sample action potentials of cardiomyocytes generated via the standard broad-spectrum Wnt inhibitor (WntC59) approach or via Sfrp2 protocol. (e) Patch-clamp data was analyzed for action potential amplitude (APA), maximal rate of depolarization (MPU) (dv/dtmax ( and action potential duration (APD) at 90% of APA. N = 15 (WntC59) or 19 (Sfrp2). Comparisons were made to the WntC59 group: *P < 0.05, ns – not significant.

Fig 5: Sfrp2-derived cardiomyocytes form gap junctions. IPSc-derived cardiomyocytes were generated via either the standard broad-spectrum Wnt inhibitor (WntC59) approach or via the Sfrp2 method. After 14 days of differentiation, the cells were analyzed. (a) Cells were delineated by staining for the cell membrane marker wheat germ agglutinin (WGA-white). Cardiomyocytes were visualized with cTnT (green) antibodies and nuclei stained with DAPI (blue). Distribution of gap junctions in cardiomyocytes was determined by immunostaining for connexin-43 (red). N = 3. Scale bar 20 microns. (b) Patch-clamp recordings (Fig. 2e) were analyzed for atrial and ventricular cardiomyocytes. (c) Cells were incubated with antibodies targeting the general cardiomyocyte marker cTNT and the ventricular-specific cardiomyocyte marker MLC2v and then subjected to FACS analysis. The graph shows the number of MLC2v + cells as a percentage of the total cardiomyocyte (cTNT +) population. N = 3. Representative traces are shown.