L11 - Enriching GABA-Positive Neuron-Biased Subpopulations from Mouse Neural Stem and Progenitor Cells Using Dielectrophoresis

Introduction: : Neural stem and progenitor cells (NSPCs) are essential for brain development and hold promise for treating neurological disorders. Understanding how progenitor subtypes give rise to neurons is key to elucidating mechanisms of differentiation. NSPC cultures are inherently heterogeneous; to address this, our lab employs label-free dielectrophoresis (DEP)-based sorting to enrich neuron-biased progenitors. DEP applies nonuniform electric fields to leverage differences in dielectric properties to separate viable cells in a non-invasive, high-throughput manner, enabling downstream functional analysis.
Using a multi-outlet microfluidic DEP sorting device designed to increase throughput and resolution, astrocyte progenitors were enriched in a single outlet. In contrast, neuron-biased progenitors were distributed across multiple outlets, suggesting greater neuronal progenitor heterogeneity. This may reflect the presence of distinct progenitor types, including those generating excitatory glutamatergic neurons from the cortex, inhibitory GABAergic neurons from the ganglionic eminence, and cortical layer-specific neurons.
To explore this heterogeneity, we sorted NSPCs isolated from embryonic day 15.5 (E15.5) cortex and E15.5 ganglionic eminence (GE) using a DEP-based hydrodynamic oblique angle parallel electrode sorter (HOAPES). Sorting outcomes were evaluated using DEP-based electrophysiological profiling. NSPC fate was assessed by differentiating sorted cells and using immunocytochemistry for differentiated neuronal markers. Together, these analyses aim to define electrophysiological and molecular features of distinct neuron progenitor populations, providing insights into lineage specification during brain development and informing future therapeutic strategies targeting NSPCs.

Materials and

Methods: : Cortical tissue and GE were dissected from E15.5 mouse telencephalon. Tissues were collected in 1× PBS with 0.6% glucose, pooled from multiple embryos (same litter), and treated as one biological replicate.

Cells were dissociated using NeuroCult and resuspended in DEP buffer (conductivity 100 µS/cm). After three washes, cells were adjusted to 1 × 10⁶ cells/mL. Viability was assessed by Trypan Blue. Sorting was conducted in which cells were collected in three outlets: Outlet 1 (O1) indicative of cells experiencing strong positive DEP force; Outlet 2 (O2) indicative of cells experiencing a weak positive DEP force; and Outlet 3 (O3), reflective of negative DEP. Sorted cells were cultured and expanded separately for post-sort analysis. Controls consisted of unsorted cells that remained in cell culture media, unsorted cells that were exposed to the electric field and experienced positive DEP, and unsorted cells that remained in the DEP buffer for the duration of cell sorting experiments. Electrophysiological properties were measured using the 3DEP system (DEPTech). 3DEP chips were sterilized, loaded with cells, and subjected to frequency sweeps (1 kHz–45 MHz, 10 Vpp, 60 s).

Neuronal differentiation and immunocytochemistry of mNSPCs: Sorted mNSPCs were expanded then transferred to coverslips. After 24 hours, differentiation media (growth factor-free) was applied for 3 days. Cells were stained with anti-TUJ1 and anti-MAP2 with Alexa Fluor secondaries. TUJ1+ and MAP2+ neurons were quantified across outlets. Co-staining with anti-GABA assessed GABAergic neurons.

Statistical tests included one-way ANOVA with Tukey post-hoc (n ≥ 3).

Results, Conclusions, and Discussions:: Results
This study investigated the heterogeneity and differentiation potential of mNSPCs using DEP-based cell sorting. The research aimed to better understand the generation of progenitors linked to different lineage fates and factors driving mNSPC differentiation. Key findings: DEP analysis using the 3DEP system successfully captured electrophysiological properties of mNSPCs and additional features including the midpoint cytoplasm frequency. The DEP characterization procedure demonstrated high reproducibility across multiple biological replicates, with only high-quality runs (R-value > 0.9) retained for analysis. DEP-based cell sorting in microfluidic devices effectively separated mNSPC subpopulations based on their dielectric properties, preserving cell viability for downstream studies. Neuronal differentiation and immunocytochemistry assays revealed the presence of GABA-positive neurons following induction in differentiation media, as evidenced by TUJ1, MAP2, and GABA staining. Statistical analysis confirmed significant differences in the differentiation potential of sorted subpopulations.

Discussion
These findings have important implications for regenerative medicine applications, where directing NSPC differentiation towards desired cell types is crucial. The ability to isolate and study specific progenitor subsets using DEP opens up new avenues for stem cell research and therapeutic development. Future studies should investigate the molecular mechanisms underlying the observed differences in dielectric properties between mNSPC subpopulations,
explore the long-term stability and functionality of DEP-sorted mNSPC subpopulations in various differentiation pathways, and assess the potential of DEP-sorted stem cells for clinical applications. The research presented here demonstrates the power of DEP-based cell sorting in unraveling NSPC heterogeneity and advancing our understanding of stem cell biology and regenerative medicine.

Conclusions
The results demonstrate that DEP-based cell sorting is an effective, label-free method for enriching specific mNSPC subpopulations with distinct lineage commitments. This approach allows for the isolation and study of progenitor subsets, providing insights into the molecular mechanisms governing NSPC fate decisions. The successful differentiation of sorted mNSPCs into neurons highlights the potential of this technique for identifying and isolating subpopulations with enhanced therapeutic potential, such as cells with greater neurogenic capacity.

Acknowledgements and/or References (Optional):: Acknowledgements:
National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number T32NS082174. Statistical support was provided by Clinical and Translational Science and Center for Statistical Counseling. I'd like to acknowledge the commitment demonstrated by my undergraduate students and overwhelming support from the School of Medicine, Department of Anatomy & Neurobiology ,my family, peers, and friends. Above all-- Philippians 4:13.