Apoptotic Find-me Signals are an Essential Driver of Stem Cell Conversion To The Cardiac Lineage

Pluripotent stem cells can be driven by manipulation of Wnt signaling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic mesoderm lineage and ultimately into functional cardiomyocytes 1-4. Strikingly, we observed that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) undergo widespread apoptosis upon Wnt activation, followed by a synchronous epithelial-mesenchymal transition (EMT). The EMT requires induction of transcription factors SNAI1/SNAI2 downstream of MESP1 expression, and double knock-out of SNAI1/2, or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis chemically or by ablation of pro-apoptotic genes also completely prevents the EMT, suppressing even the earliest events in mesoderm conversion, including EOMES, TBX6, and MESP1 induction. Conditioned medium from WNT-activated WT iPSCs overcomes the block to EMT by cells incapable of apoptosis (Apop-), suggesting the involvement of soluble factors from apoptotic cells in mesoderm conversion. Treatment with a purinergic P2Y receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signaling. ATP was sufficient to induce a partial EMT in Apop- cells treated with WNT activator. We conclude that nucleotides, in addition to acting as chemo-attractants for clearance of apoptotic cells can, unexpectedly, function as essential paracrine signals in mesoderm specification.