A single small molecule-based human embryo model reveals a requirement for V-ATPase in mammalian blastocyst cavitation

DMSO treatment upregulates key V-ATPase 2 subunits (ATP6V0A4 and ATP6V1B1), which are also enriched in the TE of human embryos. Genetic or pharmacological inhibition of V-ATPase activity disrupts lysosomal acidification, blocks intracellular vacuole formation, and impairs blastoid cavitation, whereas overexpression of V-ATPase subunits rescues this phenotype. Furthermore, genetic and pharmacological perturbations of V-ATPase function significantly compromise cavitation in both mouse and human blastocysts.

Finally, DMSO treatment induces membrane biomechanical changes characteristic of early embryonic development, suggesting a mode of action distinct from conventional small-molecule, signaling pathway- based induction strategies. This simple DMSO-based blastoid model recapitulates key aspects of human blastocyst development and reveals a conserved requirement for V- ATPase-mediated lysosomal acidification during early mammalian embryogenesis.