Efficient Generation of Definitive-like Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells...

The generation of hematopoietic stem and progenitor cells (HSPCs) with definitive features from human pluripotent stem cells (hPSCs) requires coordinated lineage specification together with a supportive hematopoietic microenvironment. Although 3D and aggregate-based systems can recapitulate endothelial and stromal niche features, their self-organizing architecture can introduce structural heterogeneity and batch-to-batch variability, complicating experimental standardization and process control. Here, we developed a chemically defined, feeder-free, lateral plate mesoderm (LPM)-directed 2D multicellular system that combines controlled hematopoietic specification with the spontaneous formation of an endothelial-stromal microenvironment. Through sequential differentiation from LPM to hemogenic endothelium, this system generated CD34⁺CD43⁺CD45⁺ HSPCs at frequencies of up to 86% across three hPSC lines within 10 days. Notably, an endothelial-stromal microenvironment formed spontaneously without aggregate formation, and disruption experiments implicated Notch and CXCL12/CXCR4 signaling in hematopoietic progenitor development. Transcriptomic benchmarking revealed HSC-associated molecular features in the generated cells while also identifying residual transcriptional differences from primary cord blood LT-HSCs. The resulting HSPCs exhibited clonogenic activity and multilineage differentiation potential, giving rise to enucleated erythrocytes, cytotoxic natural killer cells, and phenotypically identified monocyte-like and T-lineage cells, with erythroid enucleation reaching approximately 80%. In addition, day-10 suspension-derived CD34⁺ cells showed detectable bone marrow engraftment in vivo. Collectively, this study shows that key microenvironmental features can be retained within a simplified 2D multicellular system, providing a framework for studying hematopoiesis and refining hPSC-derived HSPC generation. STATEMENT OF SIGNIFICANCE: Recent three-dimensional and organoid-based approaches have highlighted the importance of multicellular microenvironments in hPSC-derived hematopoiesis, but their structural complexity can complicate culture handling and standardization. Here, we established a chemically defined, feeder-free planar system that directed lateral plate mesoderm toward hemogenic endothelium while supporting spontaneous emergence of endothelial, stromal, and hematopoietic populations. The resulting HSPCs exhibited features of definitive hematopoiesis, including clonogenic activity, multilineage differentiation, and evidence of bone marrow engraftment in vivo. Disruption experiments targeting DLL4/Notch and CXCL12/CXCR4 signaling supported a role for this multicellular microenvironment in progenitor output. This work shows that key multicellular microenvironmental features can be retained within a simplified planar culture, providing a framework for studying hematopoiesis and refining hPSC-derived HSPC generation.