Serum-free mesenchymal stem/stromal cell secretomes induce embryonic stem cell gastruloid formation and cardiomyocyte development.

We generated serum-supplemented and serum-free mouse ST2 mesenchymal stem/stromal cell conditioned medium (MSC CM) to treat mouse E14-Tg2A embryonic stem (ES) cells. Coating tissue culture plates with serum-free MSC CM promoted ES cell growth. In 3D ES cultures primed with the Wnt/β catenin agonist, CHIR99201, serum-free MSC CM induced gastruloid formation with axial elongation and TBXT (Brachyury) immunoreactivity localized in the elongation tips. Serum-supplemented MSC CM and control media did not have these effects. Extended gastruloid culture in serum-free MSC CM resulted in pleiomorphic structures that contracted rhythmically. These beating ES gastruloids were immunopositive for cardiac Troponin T. Proteomic analyses of serum-free MSC CM identified multiple target proteins that promote ES cell adhesion, growth, morphogenesis, mesodermal and cardiomyocyte differentiation, notably vascular endothelial growth factor and leukemia inhibitory factor. Hence, this study has utilised a novel model system to interrogate the secretome activity of MSCs and shown that this can generate ES cell-derived cardiomyocytes.