Protocol for generating human cortical organoids enriched in outer radial glia by guided differentiation

Area of Science:

• Neuroscience
• Developmental Biology
• Stem Cell Research

Background:

• Human pluripotent stem cell (hPSC)-derived brain organoids are crucial models for studying human brain development and disease.
• Existing protocols require refinement for increased reproducibility and complexity.
• Generating specific cell types and structures within organoids remains a challenge.

Purpose of the Study:

• To present a guided differentiation protocol for generating cortical forebrain organoids (COs) and cortico-pericyte (CP) assembloids.
• To enhance the robustness of the outer radial glia (oRG) population and expand the outer subventricular zone (oSVZ) within these organoids.
• To provide detailed methods for organoid generation, characterization, and single-cell dissociation.

Main Methods:

• Guided differentiation of hPSCs into cortical organoids.
• Co-culture of organoids with generated cortical pericytes to form CP assembloids.
• Immunostaining for characterization of organoid cellular composition and structure.
• Single-cell dissociation techniques for downstream analyses.

Main Results:

• Successful generation of hPSC-derived cortical organoids with a defined protocol.
• Creation of cortico-pericyte assembloids exhibiting a robust oRG population and expanded oSVZ.
• Established methods for organoid characterization and single-cell analysis.

Conclusions:

• The presented protocol offers a refined method for generating complex human cortical organoids and assembloids.
• This approach enhances key cellular populations relevant to cortical development.
• The protocol facilitates further research into human brain development and neurological disorders using advanced organoid models.