Differentiation of neural stem cells derived from human stem cells from apical papilla into neuronal-like cells undergoing maturation via 3D-neurospheres formation and neurogenic induction

Abstract

Objective: Human stem cells from apical papilla (hSCAPs) are a promising ectomesenchyme‑ derived cell source with neuronal differentiation potential. Under 3D-neurospheres induction, hSCAPs can be induced into neural stem cells (NSCs) and further committed toward neuronal lineages. This study aimed to demonstrate the differentiation of NSCs‑hSCAPs into neuronal‑like cells undergoing maturation through 3D-neurospheres formation and subsequent neurogenic induction. Design: The characterized hSCAPs were induced into NSCs via 3D-neurospheres formation. The intraspheroidal cells were verified for early neural stemness properties and subsequently dissociated and cultured under neurogenic induction conditions for 7 days. Neuronal differentiation was evaluated by identification of Nissl substance, immunofluorescent analysis of neuronal‑associated proteins, quantitative mRNA expression, and depolarization‑evoked intracellular Ca²⁺ imaging. Results: Following 3D neurosphere induction, hSCAPs formed clusters of intraspheroidal cells exhibiting typical NSCs characteristics, including high expression of Nestin and SOX2 and self‑reaggregation ability. After 7 days of neurogenic induction, the differentiated cells displayed distinct neuronal‑like morphologies, reduced expression of early neuronal markers (Nestin/NES and SOX2/SOX2), and increased expression of early neuronal differentiation-associated markers (Beta‑III tubulin/TUBB3) at both protein and mRNA levels. The synaptic vesicle‑associated gene (SV2A) was highly detected at the mRNA level. Furthermore, depolarization‑evoked Ca²⁺ responses after KCl stimulation were observed, indicating membrane excitability and voltage‑gated calcium channel in the differentiated cells. Conclusions: NSCs‑hSCAPs possess the capacity to generate neuronal‑like cells undergoing maturation via 3D-neurospheres formation and neurogenic induction.