Acoustic cell patterning reveals geometry- and substrate-dependent vasculogenesis and human embryo model development.

Precise spatial organization of cells is critical for engineering functional tissues and constructing human developmental models. Here, we report the development of acoustic cell patterning (ACP), an ultrasound-based method capable of rapidly generating spatial patterns of multiple cell types on both rigid and compliant substrates without requiring chemical or physical modification of the substrates. Spatially controlled co-patterning of human endothelial and stromal fibroblast cells through ACP reveals geometry- and substrate-dependent vascular morphogenesis. Applied to human pluripotent stem cells (hPSCs) within a microfluidic device, ACP forms neural tube-like structures exhibiting in vivo-like spatially patterned gene expression, including caudal emergence of HOXC10. ACP also generates a uniform array of hPSC clusters on soft substrates, forming epiblast-like tissues that undergo spontaneous BMP-dependent amniogenesis and primordial germ cell-like specification. These in vivo-like developmental features are not observed in structures derived from patterned hPSCs using microcontact printing on stiff substrates. Together, these results demonstrate the unique capabilities of ACP for engineering spatially organized multicellular systems and modeling early human development under conditions that better mimic in vivo environments.