From human dental pulp stem cells to functional cholinergic neurons: An optimized neurogenic differentiation protocol for new approach methodologies.

BACKGROUND/PURPOSE: Drug discovery is now supported by new approach methodologies (NAMs), creating a growing need for in vitro non-animal models that mimic human physiology and, most importantly, function. Secretory function in exocrine organs like salivary glands relies mostly on cholinergic innervation. This study aims to establish an effective NAMs protocol to generate a robust percentage of functional cholinergic neurons by minimizing fetal bovine serum (FBS) and optimizing essential cues like all-trans-retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF). MATERIALS AND METHODS: Human dental pulp stem cells (hDPSCs) were differentiated using a two-step media protocol: (1) ATRA (10 μM) in 10 % FBS-enriched medium for 5 days, and (2) BDNF (50 ng/ml) for 7 days. FBS-free and 1 % FBS were compared. Neuronal differentiation efficiency was determined via immunocytochemistry (β-III tubulin, Neurofilament M), Western blot and CellProfiler cell image analysis software (Broad Institute). Committed neurons were assessed for morphology (CellProfiler), viability (Calcein AM, Ethidium homodimer-1), subtype identity (cholinergic receptor muscarinic 1 (CHRM1), cholinergic receptor muscarinic 3 (CHRM3), and choline acetyltransferase (ChAT), and functional activity (glutamate assay). RESULTS: Treating hDPSCs with ATRA, followed by BDNF under 1 % FBS, resulted in higher expression of β-III tubulin and Neurofilament M, and cells exhibited lower proliferation rate, developed neuron-like morphology, and maintained high viability at endpoint. CHRM1, CHRM3, and ChAT were expressed in the committed neurons, and the latter two displayed the highest levels. Approximately 97 % of the cells were functional cholinergic neurons. CONCLUSION: A two-step protocol using ATRA with 10 % FBS for 5 days, followed by BDNF with 1 % FBS for 7 days, effectively generated a high number of cholinergic neurons.