Abstract Body: Background: Bioprinting enables fabrication of complex three-dimensional tissues with spatial control and scalability. However, most strategies rely on pre-differentiated cells with limited proliferation, tissue-forming capacity, and cellular diversity. These limitations restrict development of physiologically relevant in vitro models needed for emerging New Approach Methodologies (NAMs). We hypothesized that bioprinting undifferentiated induced pluripotent stem cells (iPSCs) while preserving pluripotency would enable diverse cellular composition and self-organization to generate structurally complex tissues.
Methods: We developed a bioink that supports iPSC viability and pluripotency after bioprinting. Pluripotent microtissues were printed in a gelatin support bath to generate defined geometries and then subjected to directed differentiation.
Results: Bioprinted iPSCs maintained ~90% viability for four days and retained proliferation (~40% Ki67⁺) and pluripotency (~80% OCT4⁺), comparable to 2D culture. Printed pluripotent microtissues differentiated into all three germ layers. We demonstrated fabrication of defined geometries including a primitive heart tube. Cardiac differentiation of tube-shaped constructs produced a D30 heart tube comprising multiple cardiac cell types (cardiomyocytes, fibroblasts, endothelial, and epicardial cells) and exhibiting trabeculae-like morphology, immature sarcomeres, and angiogenic endothelial networks. Constructs displayed synchronized Ca²⁺ transients. For scalable screening, bioprinted pluripotent tissue-derived cardiac organoids (BPCO) were generated in 96-well format. Compared with conventional cardiac organoids (CO), BPCO showed lower baseline beating rates (60 vs 95 bpm, P<0.0001). Both models increased beating rate with isoproterenol, whereas BPCO showed reduced sensitivity to nifedipine.
Conclusions:
Undifferentiated iPSC bioprinting enables programmable tissue geometry, developmental self-organization, and scalable organoid production. This developmental bioprinting strategy provides a platform for engineering more physiological human heart models and supports development of NAMs-based drug discovery and safety testing.