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

Area of Science:

• Biotechnology
• Stem Cell Biology
• Synthetic Biology

Background:

• Human induced pluripotent stem cell (hiPSC) technology is crucial for studying human biology and disease.
• Existing methods have limitations in precisely modifying proteins within hiPSC-derived models.

Purpose of the Study:

• To engineer hiPSCs with an expanded genetic code for site-specific non-canonical amino acid (ncAA) incorporation.
• To demonstrate the utility of this technology in neural stem cells, neurons, and brain organoids.

Main Methods:

• PiggyBac-mediated transgenesis was used to integrate expression cassettes for pyrrolysyl-tRNA synthetase (PylRS) and pyrrolysyl-tRNA (PylT).
• Engineered hiPSCs were differentiated into neural cell types and brain organoids.
• Site-specific incorporation of ncAAs was achieved in response to amber stop codons.

Main Results:

• Engineered hiPSCs and their derivatives maintained the amber suppression machinery.
• ncAA-bearing reporter proteins were successfully produced in these cells and organoids.
• The incorporated ncAA served as a bioorthogonal handle for fluorescent dye labeling.

Conclusions:

• Genomic integration of PylRS/PylT enables site-specific ncAA mutagenesis in hiPSC-derived models.
• This approach provides a versatile tool for probing and manipulating proteins in complex human tissue models.
• Expanded genetic code in hiPSCs opens new avenues for disease research and therapeutic development.