Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Engineered nerve guidance conduits (NGCs) have been demonstrated for repairing peripheral nerve injuries. However, there remains a need for an advanced biofabrication system to build NGCs with complex architectures, tunable material properties, and customizable geometrical control. Here, a rapid continuous 3D-printing platform was developed to print customizable NGCs with unprecedented resolution, speed, flexibility, and scalability. A variety of NGC designs varying in complexity and size were created including a life-size biomimetic branched human facial NGC. In vivo implantation of NGCs with microchannels into complete sciatic nerve transections of mouse models demonstrated the effective directional guidance of regenerating sciatic nerves via branching into the microchannels and extending toward the distal end of the injury site. Histological staining and immunostaining further confirmed the progressive directional nerve regeneration and branching behavior across the entire NGC length. Observational and functional tests, including the von Frey threshold test and thermal test, showed promising recovery of motor function and sensation in the ipsilateral limbs grafted with the 3D-printed NGCs.