Precardiac Organoids Form Two Heart Fields via Bmp/Wnt Signaling

The discovery of the first heart field (FHF) and the second heart field (SHF) led us to understand how cardiac lineages and structures arise during development. However, it remains unknown how they are specified. Here, we generate precardiac spheroids with pluripotent stem cells (PSCs) harboring GFP/RFP reporters under the control of FHF/SHF markers, respectively. GFP+ cells and RFP+ cells appear from two distinct areas and develop in a complementary fashion. Transcriptome analysis shows a high degree of similarities with embryonic FHF/SHF cells. Bmp and Wnt are among the most differentially regulated pathways, and gain- and loss-of-function studies reveal that Bmp specifies GFP+ cells and RFP+ cells via the Bmp/Smad pathway and Wnt signaling, respectively. FHF/SHF cells can be isolated without reporters by the surface protein Cxcr4. This study provides novel insights into understanding the specification of two cardiac origins, which can be leveraged for PSC-based modeling of heart field/chamber-specific disease. We sought to determine the cellular identities of PSC-derived GFP+ and RFP+ cells. To do this, GFP+ and RFP+ CPCs were FACS-isolated from the spheroids at day 5.5 or from Hcn4GFP; Tbx1Cre; Ai9 mouse embryos at E7.75 and subjected to RNA-sequencing.