Engineered Human Induced Pluripotent Cells Enable Genetic Code Expansion in Brain Organoids

Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac‐mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl‐tRNA synthetase (PylRS), pyrrolysyl‐tRNA (PylT) and the target protein of interest enables site‐specific incorporation of a non‐canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA‐bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site‐directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC‐derived cell types and complex tissue models.