A Triple‐Fusion Protein Probe for Multimodal Imaging of Neural Lineage Cells In Vitro and After Transplantation in Brain

Abstract Imaging the behavior and fate of implanted cells is crucial in cell therapy. However, it remains challenging to achieve synchronized tracing of different behaviors for the same cell due to the lack of a multimodal imaging probe. Herein, through flexible polypeptide linkers, S‐L‐D‐S and G‐S‐S‐G, a multifunctional probe is constructed by fusing three imaging proteins, including bioluminescent imaging reporter (NanoLuc), fluorescent protein (mRuby2), and calcium indicator (GCamp6f). A lentivirus carrying the triple‐fusion probe is prepared for stable transfection of human induced pluripotent stem cells (hiPSCs). During the long‐term differentiation of hiPSCs into neural lineage cells and human cerebral organoids (hCOs), the multifunctional probe could be used for bioluminescent imaging with ≈10‐cell sensitivity, single‐cell fluorescent imaging, and functional imaging of calcium activity. Then, hiPSC‐derived neural progenitors and hCOs are transplanted into the mouse brains. Multimodal imaging is performed to trace the survival, distribution, integration, and functional activity of transplanted cells, providing multi‐scale data of exogenous cell behavior and fate in host brains. These results demonstrate that the triple‐fusion probe could be a powerful tool for the comprehensive evaluation of cell therapy.