Human Organoids Reveal PTEN-Driven Mesendoderm Specification via Retinoic Acid Signaling Suppression

During early embryogenesis, epiblast cells ingress through the primitive streak (PS) to adopt a mesendoderm (MES) fate, giving rise to both mesodermal and endodermal lineages. Robust systems that accurately recapitulate MES specification-and thereby enable a comprehensive understanding of cell fate transitions during PS formation-remain limited. Here, we describe 3-dimensional MES organoids derived from human induced pluripotent stem cells that faithfully model in vivo human MES specification, particularly from the anterior PS. MES specification requires PTEN expression, as PTEN loss profoundly impairs mesoderm and definitive endoderm derivatives. Integrative multi-omics analyses-including transcriptomics, chromatin accessibility, metabolomics, and phosphoproteomics-on wild-type and PTEN^-/- MES cells revealed that PTEN suppresses retinoic acid (RA) signaling during MES commitment. Furthermore, we identified the RA-degrading enzyme CYP26A1 as a downstream effector of PTEN. Notably, while excessive RA induced by PTEN ablation is detrimental to MES cell generation, physiological levels of RA are necessary. Collectively, the human MES organoids offer a valuable model for dissecting early human development, and our findings identify PTEN as a key regulator of MES fate commitment through inhibition of RA signaling.