Generation of 3D Hemogenic Gastruloids From Mouse Embryonic Stem Cells

Embryonic blood formation encompasses the independent generation of different cell types in distinct cellular and anatomical environments, reflecting highly coordinated specific hierarchies of interacting tissues. Despite widespread use of embryonic stem cells (ESC) and induced pluripotent stem cell (iPSC)-based models to attempt to capture blood development in vitro and generate hematopoietic stem cells (HSC), a system that fully captures the spatial and temporal complexity of embryonic hematopoiesis is still lacking. In recent years, gastruloid models have emerged as powerful representations of early development, demonstrating self-organizing behaviors such as symmetry breaking, elongation, multi-axis formation, somitogenesis, and early organogenesis, with striking parallels to embryonic processes. Here, we present a protocol to generate hemogenic gastruloids (haemGx) from mouse ESC (mESC) that closely recapitulates the multi-stage, multi-niche process of blood formation and generates developmentally accurate hematopoietic progenitors. The haemGx model has been proven valuable in understanding embryonic hematopoiesis, as well as an in vitro model of forms of infant leukemia with an embryonic, in utero origin. Key features • The haemGx protocol allows the generation of developmentally accurate endothelial and hematopoietic precursor and progenitor cell types. • The haemGx protocol achieves a level of spatiotemporal control that closely recapitulates key aspects of embryonic development. • The haemGx protocol is compatible with multiple mESC lines, enabling reproducible generation of developmental blood cell types across different genetic backgrounds. • At endpoint, individual haemGx reaches ~800-1,000 μm in diameter and contains 30,000/50,000 cells. The 96-well format allows for upscaling and high-throughput applications.