Protocol for isolating nuclei from human stem cell-derived grafts for single-nucleus RNA sequencing

Area of Science:

• Neuroscience
• Stem Cell Biology
• Molecular Biology

Background:

• Single-nucleus RNA sequencing (snRNA-seq) offers high-resolution transcriptomic analysis of brain tissue.
• Understanding cell identity is crucial for studying neurodegeneration and neural repair.
• Human stem cell grafts are promising for brain repair but require methods to assess their integration and function.

Purpose of the Study:

• To present a detailed protocol for isolating nuclei from human stem cell-derived grafts within the rat brain.
• To enable high-resolution transcriptomic analysis of these grafts in vivo.
• To facilitate the assessment of graft composition, integration, and neuronal identity.

Main Methods:

• Protocol involves vibratome sectioning of the rat brain.
• Graft dissection and subsequent nuclear extraction are key steps.
• Fluorescence-activated sorting is employed for nuclear isolation.

Main Results:

• The presented workflow successfully isolates nuclei from human stem cell grafts in the rat brain.
• This method is suitable for analyzing human neurons within host tissue.
• It supports the assessment of neurons sensitive to dissociation procedures.

Conclusions:

• This protocol provides a robust method for analyzing human stem cell grafts in vivo using snRNA-seq.
• It enhances the study of neurodegeneration and repair by enabling detailed analysis of graft components.
• The workflow supports comprehensive evaluation of graft integration and neuronal function in the living brain.