A conserved bioelectrical signature defines subventricular zone-derived human fetal neural stem cells and tracks their differentiation state

Roberta De Zio^1*Diletta Lucia Capobianco¹Daniela Celeste Profico²Giada D'Aloisio³Giuseppe Procino¹Maurizio Gelati⁴Angelo Luigi Vescovi^5,6Francesco Pisani¹Maria Svelto¹Andrea Gerbino^1*

• ¹Department of Biosciences, Biotechnologies and Environment, University of Bari Aldo Moro, Bari, Italy
• ²ProPharma LLC, Leiden, Netherlands
• ³IRCSS Casa Sollievo della Sofferenza, Unità Produttiva per Terapie Avanzate, San Giovanni Rotondo, Italy
• ⁴Fondazione IRCSS Istituto Neurologico “C.Besta”, Milan, Italy
• ⁵Faculty of Medicine, Link Campus University, Rome, Italy
• ⁶Abu Dhabi Stem Cell Centre, Abu Dhabi, United Arab Emirates

Introduction: Human fetal neural stem cells (hfNSCs) from the subventricular zone (SVZ) are employed in clinical trials for neurodegenerative diseases, yet their bioelectrical properties remain largely unexplored. Molecular markers alone do not reliably correlate with functional state, highlighting the need for complementary functional descriptors. Methods: We performed whole-cell patch-clamp recordings on hfNSCs from three independent SVZ donors (15–16 weeks of gestation) to characterize resting membrane potential (Vm) and voltage-gated currents, assessing inter-donor reproducibility and differentiation dynamics over 30 days in vitro. Results: hfNSCs exhibited a highly reproducible bioelectrical signature across all donors, characterized by a depolarized resting membrane potential (~ −30 mV), a non-excitable profile, and a stereotyped composition of outward K+ currents. The two current components were resolved using combined biophysical and pharmacological approaches, while Western blot analysis confirmed the expression of Kv4.2 and Kv1.1 channel subtypes functionally consistent with IA and IK, respectively. Remarkably, inter-donor variability in bioelectrical parameters was minimal despite independent cell line derivation. During differentiation, Vm underwent rapid hyperpolarization within 24 hours, representing the earliest detectable functional transition. IA showed progressive reduction detectable as early as 24 hours and more pronounced by day 15, while IK remained stable throughout. By day 30, inward voltage-gated currents emerged in approximately 60% of cells, consistent with progression toward more differentiated neuro-glial electrophysiological states; however, cells remained non-excitable under our recording conditions. This late-stage divergence highlights heterogeneity in maturation trajectories and completes a temporally ordered sequence of electrical remodeling. Conclusions: SVZ-derived hfNSCs possess a reproducible bioelectrical signature within the 15–16-week gestational window across independent donors, supporting electrophysiological profiling as a quantitative functional benchmark for identity and standardization. During in vitro differentiation, Vm hyperpolarization and IA/IK remodeling track early functional progression, whereas the late emergence of inward currents in only a subset of cells indicates increased heterogeneity at later stages. Overall, these findings support bioelectrical profiling as a quantitative functional biomarker with potential utility for standardization and quality assessment in hfNSC-based regenerative therapies.