CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects

•We examined differentiation of embryonic stem cells that lacked Cstf2 (CstF-64)•Cstf2-null ESCs could differentiate into mesoderm and ectoderm, but not endoderm•Cstf2-null cells could not be induced to differentiate into cardiomyocytes•Factors from XEN cells were able to restore cardiomyocyte differentiation•We propose that CstF-64 has a role in regulating cardiac specification

Although adult cardiomyocytes have the capacity for cellular regeneration, they are unable to fully repair severely injured hearts. The use of embryonic stem cell (ESC)-derived cardiomyocytes as transplantable heart muscle cells has been proposed as a solution, but is limited by the lack of understanding of the developmental pathways leading to specification of cardiac progenitors. Identification of these pathways will enhance the ability to differentiate cardiomyocytes into a clinical source of transplantable cells. Here, we show that the mRNA 3′ end processing protein, CstF-64, is essential for cardiomyocyte differentiation in mouse ESCs. Loss of CstF-64 in mouse ESCs results in loss of differentiation potential  toward the endodermal lineage. However, CstF-64 knockout (Cstf2E6) cells were able to differentiate into neuronal progenitors, demonstrating that some differentiation pathways were still intact. Markers for mesodermal differentiation were also present, although Cstf2E6 cells were defective in forming beating cardiomyocytes and expressing cardiac specific markers. Since the extraembryonic endoderm is needed for cardiomyocyte differentiation and endodermal markers were decreased, we hypothesized that endodermal factors were required for efficient cardiomyocyte formation in the Cstf2E6 cells. Using conditioned medium from the extraembryonic endodermal (XEN) stem cell line we were able to restore cardiomyocyte differentiation in Cstf2E6 cells, suggesting that CstF-64 has a role in regulating endoderm differentiation that is necessary for cardiac specification and that extraembryonic endoderm signaling is essential for cardiomyocyte development.