Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system

Area of Science:

• Neuroscience
• Stem Cell Biology
• Biotechnology

Background:

• Oligodendrocytes (OLs) are crucial for myelin formation in the central nervous system (CNS).
• Modeling human oligodendrocyte differentiation and axon myelination in vitro remains challenging.
• Brain microphysiological systems (bMPS) offer a more in vivo-like environment for studying myelination.

Purpose of the Study:

• To generate a human induced pluripotent stem cell (hiPSC) line for tracking oligodendrocytes using a PLP1-sfGFP reporter.
• To validate and utilize this reporter line within a 3D bMPS model for studying oligodendrocyte development and myelination.
• To assess the model's response to chemical stressors like cuprizone.

Main Methods:

• CRISPR/Cas9 technology was employed to tag proteolipid protein 1 (PLP1) with super-fold GFP (sfGFP) in hiPSCs.
• Reverse transfection yielded higher Knock-In (KI) efficiency for the PLP1-sfGFP reporter compared to forward transfection.
• Validated PLP1-sfGFP hiPSC clones were differentiated into bMPS, and oligodendrocyte differentiation stages were tracked via GFP expression and morphology.

Main Results:

• Successful generation and validation of a PLP1-sfGFP hiPSC reporter line.
• Demonstrated tracking of oligodendrocyte differentiation, migration, and myelination in 3D bMPS.
• Quantified changes in PLP1-sfGFP expression and intensity upon cuprizone challenge, indicating model responsiveness.

Conclusions:

• This study presents an efficient method for generating hiPSC KI lines.
• A novel 3D bMPS model is described for studying oligodendrocyte biology in vitro.
• The model facilitates research into neurodevelopment, chemical toxicology, and disease-associated stressors affecting myelination.