This study explored whether a hypoxic environment could enhance the neuronal differentiation of olfactory mucosa mesenchymal stem cells (OM-MSCs) when cultured with olfactory ensheathing cell (OEC) supernatant, and sought to elucidate the underlying mechanisms involved. Initially, both OM-MSCs and OECs were isolated and cultured, and their identities were confirmed using flow cytometry and immunofluorescence techniques.

The OM-MSCs were then divided into three experimental groups: one group (Group A) was cultured under hypoxic conditions (3% O₂) with the addition of a hypoxia-inducible factor-1α (HIF-1α) inhibitor (YC-1) alongside OEC supernatant; a second group (Group B) was cultured under the same hypoxic conditions (3% O₂) with OEC supernatant but without the inhibitor; and a control group was maintained under normoxic conditions (21% O₂) with OEC supernatant.

Neuronal differentiation was assessed by examining the expression of βIII-tubulin, a neuronal marker, through immunofluorescence. Quantitative polymerase chain reaction (qPCR) and Western blot analyses were performed to measure the mRNA and protein levels of HIF-1α, βIII-tubulin, and glial fibrillary acidic protein (GFAP), a marker for neuroglial cells. In addition, the functional maturity of the differentiated neurons was evaluated by analyzing potassium channel activity using the patch clamp technique.

The results showed that the neurons differentiated from OM-MSCs under hypoxic conditions with OEC supernatant (Group B) exhibited the highest levels of βIII-tubulin expression. qPCR analysis revealed that HIF-1α expression was significantly higher in Group B compared to the other groups, suggesting that hypoxia-induced activation of HIF-1α plays an important role in this differentiation process. Western blot results corroborated these findings by demonstrating significantly elevated βIII-tubulin protein levels and a marked reduction in GFAP expression in Group B. Furthermore, the patch clamp recordings confirmed that the potassium channels in these neurons were activated, indicative of functional neuronal properties.

In conclusion, the study provides compelling evidence that hypoxic conditions markedly enhance the neuronal differentiation of OM-MSCs when combined with OEC supernatant, while concurrently reducing the generation of neuroglial cells. This enhanced differentiation appears to be closely associated with the activation of the HIF-1 signaling pathway, highlighting a potential strategy for optimizing stem cell-based neuronal regeneration under hypoxic conditions. Lificiguat