biostudies-literatureUnknown18(2)Zhang JMacrophages armed with chimeric antigen receptors (CARs) provide a potent new option for treating solid tumors. However, genetic engineering and scalable production of somatic macrophages remains significant challenges. Here, we used CRISPR-Cas9 gene editing methods to integrate an anti-GD2 CAR into the AAVS1 locus of human pluripotent stem cells (hPSCs). We then established a serum- and feeder-free differentiation protocol for generating CAR macrophages (CAR-Ms) through arterial endothelial-to-hematopoietic transition (EHT). CAR-M produced by this method displayed a potent cytotoxic activity against GD2-expressing neuroblastoma and melanoma in vitro and neuroblastoma in vivo. This study provides a new platform for the efficient generation of off-the-shelf CAR-Ms for antitumor immunotherapy.Stem cell reports585-596https://www.ebi.ac.uk/biostudies/studies/S-EPMC9968983biostudies-literatureGeneration of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies.PMC9968983Swanson SZhang JBolin JBernstein MNSteill JStewart RForsberg MHThomson JAMajumder ACapitini CMSlukvin IIDuffin BWebster SBrown MEfalseGeneration of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies.Macrophages armed with chimeric antigen receptors (CARs) provide a potent new option for treating solid tumors. However, genetic engineering and scalable production of somatic macrophages remains significant challenges. Here, we used CRISPR-Cas9 gene editing methods to integrate an anti-GD2 CAR into the AAVS1 locus of human pluripotent stem cells (hPSCs). We then established a serum- and feeder-free differentiation protocol for generating CAR macrophages (CAR-Ms) through arterial endothelial-to-hematopoietic transition (EHT). CAR-M produced by this method displayed a potent cytotoxic activity against GD2-expressing neuroblastoma and melanoma in vitro and neuroblastoma in vivo. This study provides a new platform for the efficient generation of off-the-shelf CAR-Ms for antitumor immunotherapy.2023-01-01T00:00:00Z2023 Feb2025-04-18T14:22:16.876Z2025-04-07T00:30:37.491ZS-EPMC99689833663878810.1016/j.stemcr.2022.12.012