An Optogenetic-Controlled Cell Reprogramming System for Driving Cell Fate and Light-Responsive Chimeric Mice.

Area of Science:

• Stem cell biology
• Gene editing technologies
• Optogenetics

Background:

• Pluripotent stem cells (PSCs) are crucial for regenerative medicine, disease modeling, and drug discovery.
• Traditional induced PSCs (iPSCs) generation via transcription factor (TF) overexpression has limitations, including side effects and unpredictable targeting.
• Precise control over endogenous TF expression is needed for effective cell fate reprogramming.

Purpose of the Study:

• To develop a novel light-inducible cell reprogramming (LIRE) system for precise control over endogenous gene expression.
• To generate induced pluripotent stem cells (iPSCs) using optogenetic regulation of key pluripotency genes (Sox2 and Oct4).
• To demonstrate the utility of the LIRE system for in vivo gene control and genetic screening in mice.

Main Methods:

• Development of a LIRE system combining a cryptochrome photoreceptor with CRISPR/Cas9 nuclease-deficient technology.
• Optogenetic regulation of endogenous Sox2 and Oct4 loci using light-emitting diode illumination to reprogram mouse embryonic fibroblasts into iPSCs (iPSC_LIRE).
• Differentiation of iPSC_LIRE cells and generation of optogenetic chimeric mice via blastocyst injection.

Main Results:

• Successful generation of iPSC_LIRE cells from mouse embryonic fibroblasts through light-induced reprogramming.
• Demonstrated efficient differentiation of iPSC_LIRE cells into various cell types.
• Successfully generated optogenetic chimeric mice, enabling non-invasive, in vivo control of endogenous genes.

Conclusions:

• The LIRE system provides a remote, traceless, and non-invasive method for cellular reprogramming.
• This technology offers a valuable tool for controlled gene expression studies and genetic screens in mice.
• The LIRE system has significant potential for basic research, disease modeling, and regenerative medicine applications.