Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis.

INTRODUCTION: Stem cell-based dental pulp regeneration has been extensively studied, mainly focusing on exploiting dental stem cells' osteogenic and angiogenic potentials. Dental stem cells' neurogenic role is often overlooked. Stem cells from apical papilla (SCAPs), originating from the neural crest and capable of sphere formation, display potent neurogenic capacity. This study aimed to investigate the interactions of neuronally induced stem cells from apical papilla (iSCAP) spheres, SCAPs, and human umbilical vascular endothelial cells (HUVECs) on vasculogenesis and neurogenesis. METHODS: SCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small molecule neural induction medium, and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP sphere interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with ß-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. RESULTS: Neural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, ß-tubulin III, microtubule-associated protein 2, neuron-specific enolase, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared with iSCAPs. iSCAP sphere, HUVEC, and SCAP direct coculture significantly enhanced vessel formation along with up-regulated VEGF (P < .001) and multiple neural markers, such as Nestin (P < .01), microtubule-associated protein 2 (P < .001), S100 (P < .001), and NG2 (P < .001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and alpha-smooth muscle actin-positive and CD31-positive cells assembling for the vascular constitution. CONCLUSIONS: iSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.

Transplantation of Neural Progenitor Cells Derived from Stem Cells from Apical Papilla Through Small-Molecule Induction in a Rat Model of Sciatic Nerve Injury.

BACKGROUND: Stem cell-based transplantation therapy holds promise for peripheral nerve injury treatment, but adult availability is limited. A cell culture protocol utilizing a small-molecule cocktail effectively reprogrammed stem cells from apical papilla (SCAPs) into neural progenitor cells, subsequently differentiating into neuron-like cells. This study aims to evaluate neural-induced SCAPs, with and without small-molecule cocktail, for sciatic nerve repair potential. METHODS: A scaffold-free cell sheet technique was used to construct a three-dimensional cell sheet. Subsequently, this cell sheet was carefully rolled into a tube and seamlessly inserted into a collagen conduit, which was then transplanted into a 5 mm sciatic nerve injury rat model. Functional sciatic nerve regeneration was evaluated via toe spread test, walking track analysis and gastrocnemius muscle weight. Additionally, degree of sciatic nerve regeneration was determined based on total amount of myelinated fibers. RESULTS: Small-molecule cocktail induced SCAPs enhanced motor function recovery, evident in improved sciatic function index and gastrocnemius muscle retention. We also observed better host myelinated fiber retention than undifferentiated SCAPs or neural-induced SCAPs without small-molecule cocktail. However, clusters of neuron-like cell bodies (surrounded by sparse myelinated fibers) were found in all cell sheet-implanted groups in the implantation region. This suggests that while the implanted cells likely survived transplantation, integration was poor and would likely hinder long-term recovery by occupying the space needed for host nerve fibers to project through. CONCLUSION: Neural-induced SCAPs with small-molecule cocktail demonstrated promising benefits for nerve repair; further research is needed to improve its integration and optimize its potential for long-term recovery.