Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence

Area of Science:

• Neuroscience
• Stem Cell Biology
• Cellular Biology

Background:

• Adult neurogenesis involves neural stem cells (NSCs) producing new neurons.
• NSCs exist in a quiescent (G0) state, exiting the cell cycle but remaining responsive.
• Understanding NSC quiescence and activation is crucial for neurogenesis research, but identification methods are limited.

Purpose of the Study:

• To develop a novel, label-free protocol for identifying and enriching quiescent NSCs (qNSCs) and activated NSCs (aNSCs) in vitro.
• To provide a toolkit for studying NSC quiescence and activation dynamics at single-cell resolution.

Main Methods:

• Utilizing confocal microscopy to identify autofluorescent markers for classifying NSC activation state based on intensity.
• Employing fluorescence-activated cell sorting (FACS) for NSC classification and enrichment using autofluorescence.
• Applying multiphoton fluorescence lifetime imaging (FLIM) for single-cell resolution classification and tracking quiescent exit dynamics.

Main Results:

• Demonstrated a method to classify NSC activation state using autofluorescence intensity via confocal microscopy and FACS.
• Showcased FLIM's capability for single-cell resolution classification and tracking of quiescent exit dynamics.
• Established a live-cell, label-free toolkit for studying NSC quiescence and activation.

Conclusions:

• The developed protocol offers a powerful, label-free approach to study neural stem cell quiescence and activation.
• This toolkit enables precise identification and enrichment of qNSCs and aNSCs, advancing neurogenesis research.
• The methods provide new avenues for investigating the dynamic processes of NSC exit from quiescence.