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

Here, we describe a simple and robust system in which dimethyl sulfoxide (DMSO) alone induces blastoid formation from human nPSCs. This model recapitulates key pre- and post-implantation features and exhibits enhanced polar trophectoderm (TE) organization, more efficient attachment within an implantation-relevant window, improved epiblast lumenogenesis associated with amniotic cavity formation, and more robust, sustained expansion of embryonic lineages following attachment. Using this system, we reveal a previously unrecognized mechanism underlying TE cavitation and identify lysosome-associated genes — particularly subunits of the proton pump V-ATPase — as essential regulators of blastoid 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.