Abstract 15547: Sfrp2 Mediates Ipsc to Cardiomyocyte Differentiation via Competing Actions on Wnts

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Induced pluripotent stem (iPS) cells provide a novel avenue to investigate how precursors differentiate into cardiomyocytes. In this study we examined our hypothesis that iPS differentiation is regulated by a balance between cardiogenic and inhibitory Wnt proteins. Recently, we have discovered that iPS cells differentiate into cardiomyocytes in response to Sfrp2 (control: 0% cardiomyocytes; Sfrp2 treated: 25% cardiomyocytes. N=3. P<0.01). Since Sfrp2 mediates its effects by binding to Wnt proteins, we hypothesized that Sfrp2 promotes cardiomyocyte differentiation by binding to a Wnt that prevents iPS cells from differentiating into cardiomyocytes. To identify the inhibitory Wnt protein, we studied the effects of Wnt blocking antibodies on iPS and differentiation into cardiomyocytes. We observed that in control iPS cells or iPS cells incubated with a Wnt5 blocking antibody no cardiomyocytes appeared. In contrast, iPS cells incubated with a Wnt3a blocking antibody spontaneously and robustly differentiated into cardiomyocytes (40%, N=5, P<0.001). Co-immunoprecipitation experiments demonstrated that Sfrp2 binds to Wnt3a (N=3, P<0.001). Furthermore, Sfrp2 binding to Wnt3a inhibited b-catenin activation in iPS cells (80% active b-catenin in control cells; 10% active b-catenin in Sfrp2 and Wnt3a blocking antibody treated cells. N=3. P<0.05). To identify the cardiogenic Wnt, we again screened iPS differentiation following incubation with various Wnt blocking antibodies. Through this process, we found that the positive effects of Sfrp2 or Wnt3a blocking antibodies were completely lost when the iPS were co-cultured with Wnt11 blocking antibodies (Wnt3a blocking antibody alone: 40% cardiomyocytes; Wnt3a plus Wnt11 blocking antibody: 0% cardiomyocytes). Interestingly, in iPS cells incubated with Wnt11 blocking antibody, b-catenin was re-activated (control cells: 10% active b-catenin; cells incubated with Wnt11 blocking antibody: 60% active b-catenin. N=3. P<0.05). In summary, iPS cells do not spontaneously differentiate into cardiomyocytes because of opposing effects of Wnt3a and Wnt11. Sfrp2 promotes cardiomyocyte differentiation by binding to Wnt3a, allowing Wnt11 to induce iPS cells to differentiate into cardiomyocytes.