Porous microscaffold enables spontaneous hematopoietic cell differentiation and continuous macrophage production from...

Macrophages play critical roles in various physiological and pathological processes, yet their scalable production for therapeutic applications remains a challenge. Here, we present a porous microscaffold (PMS) that enables robust hematopoietic differentiation from human pluripotent stem cell (hPSC)-derived mesoderm. Within PMS, mesoderm cells self-organize into vascular structures reminiscent of the early aorta-gonad-mesonephros (AGM) niches, promoting efficient endothelial-to-hematopoietic transition with minimal cytokine supplementation. Additionally, endothelial-cell-modified PMS enriches paracrine signals and activates key genes involved in macrophage proliferation and self-renewal. Multi-omics analyses delineate the gene network driving enhanced macrophage expansion within this 3D microenvironment. Functionally, PMS-derived macrophages exhibit potent therapeutic potential by effectively alleviating drug-resistant Streptococcus pneumoniae infections in mice. These results establish PMS as a cost-effective and scalable platform for producing functional macrophages, paving the way for off-the-shelf cell-based therapies.