Abstract
Background
Diabetic peripheral neuropathy (DPN) lacks effective disease-modifying therapies, partly due to the limited availability of standardized human-relevant in vitro models. Human pluripotent stem cell (hPSC)-derived sensory neurons provide a potential platform for investigating human neuronal vulnerability under diabetes-associated stress conditions and evaluating neuroprotective strategies.New method
In this study, we established an hPSC-derived sensory neuron-enriched platform to model diabetes-associated neuronal stress. Human PSCs were differentiated into sensory neuron-enriched cultures characterized by sensory neuronal markers BRN3A (POU4F1), ISL1, and Peripherin (PRPH), as well as nociceptive-associated markers including TRPV1 and Nav1.7 (SCN9A). A sequential metabolic-oxidative stress paradigm was developed using high glucose (HG, 45mM) pretreatment followed by hydrogen peroxide (H₂O₂)-induced oxidative challenge, with mannitol osmotic controls incorporated.Results
High glucose exposure reduced cellular viability, whereas osmotic control conditions produced minimal effects. Combined HG/H₂O₂ exposure induced greater cellular injury and oxidative alterations than individual stress conditions. Pharmacological intervention with N-acetylcysteine (NAC), minocycline, or metformin partially restored cellular viability, reduced oxidative signals, and improved mitochondrial membrane potential-associated changes. Long-term morphological analysis demonstrated that early intervention preserved neurite network integrity and attenuated persistent reductions in neurite length following metabolic-oxidative stress.Comparison with existing methods
Compared with conventional in vitro models based primarily on non-human systems or high glucose exposure alone, this hPSC-derived sensory neuron platform provides a human-relevant approach that incorporates sequential metabolic and oxidative challenges and enables evaluation of both acute cellular responses and long-term neuronal structural alterations.Conclusions
This study establishes a human sensory neuron-based experimental system for investigating diabetes-associated neuronal vulnerability and evaluating potential neuroprotective compounds. The platform provides a complementary tool for studying metabolic-oxidative mechanisms underlying sensory neuronal injury associated with DPN.