A BSTRACT Background: Stem cells, known for their self-renewal and differentiation capabilities, exhibit unique metabolic characteristics that play a crucial role in cellular differentiation. In this study, we investigated the metabolic changes of Wharton’s jelly mesenchymal stem cells (MSCs) during early neuronal differentiation, focusing on key metabolites such as lactate, pyruvate, citrate, and acetyl coenzyme A (acetyl-CoA). Wharton’s jelly MSCs were induced to neuronal differentiation using N2, EGF, bFGF, B27, and forskolin. These findings reveal significant metabolic reprogramming, characterized by a shift from glycolysis toward oxidative metabolism, evidenced by decreased lactate production and modulated pyruvate and citrate levels. The lactate/pyruvate (L/P) ratio changes and acetyl-CoA levels indicate a dynamic adjustment in metabolic pathways supporting the transition from stem cell-like states to neuroprogenitor cells. Immunofluorescence analysis confirmed the expression of neuronal markers (β3-tubulin and NeuN) by days 7 and 14, validating the progression to immature and mature neuronal states. This study highlights the metabolic plasticity of Wharton’s jelly MSCs in neurogenesis. It reveals metabolite changes that could demonstrate neuronal maturation, providing important insights for optimizing metabolic conditions in future regenerative medicine applications. Context: Although the neuronal differentiation potential of Wharton’s jelly-derived MSCs (WJ-MSCs) has been well established, the associated metabolic dynamics remain poorly understood. Previous studies have suggested that a metabolic shift from glycolysis to oxidative phosphorylation is essential for neuronal maturation, yet specific alterations in key metabolites such as lactate, pyruvate, citrate, and acetyl-CoA during this transition are not well characterized in WJ-MSCs. This study addresses this gap by investigating the temporal metabolic reprogramming events during early neurogenesis. It provides insights into the bioenergetic basis of stem cell-derived neuronal differentiation and its relevance for optimizing neuroregenerative strategies. Aims: This study aims to investigate the metabolic changes occurring during the early stages of neuronal differentiation of Wharton’s jelly-derived MSCs (WJ-MSCs). Specifically, it focuses on key metabolites – lactate, pyruvate, citrate, and acetyl-CoA – to elucidate how energy metabolism is reprogrammed as WJ-MSCs transition into neuroprogenitor cells. Settings and Design: This in vitro experimental study was conducted using previously isolated and cryopreserved human Wharton’s jelly-derived MSCs (WJ-MSCs). It was designed to induce neuronal differentiation over a 14-day protocol and evaluate associated temporal changes in metabolic profiles during the early stages of neurogenesis. Subjects and Methods: Human Wharton’s jelly-derived MSCs (WJ-MSCs) previously characterized and cryopreserved were thawed and cultured in Dulbecco’s Modified Eagle Medium/F12 medium. Neuronal differentiation was induced using a sequential protocol including N2, EGF, bFGF, B27, and forskolin over 14 days. Metabolic changes were analyzed on days 2, 5, and 7 by measuring lactate, pyruvate, citrate, and acetyl-CoA levels using colorimetric and fluorometric assay kits. Immunofluorescence staining for β3-tubulin and NeuN was performed to confirm neuronal marker expression. All experiments were repeated 3–9 times using cells derived from six donors. Statistical Analysis Used: All statistical analyses were performed using SPSS version 29.0. Data were presented as mean ± standard error of the mean. The Mann–Whitney U -test was applied to compare differences between the groups. P < 0.05 was considered statistically significant. Results: Immunofluorescence staining confirmed neuronal differentiation of WJ-MSCs, showing β3-tubulin expression on day 7 and NeuN expression on day 14. Metabolite analysis revealed a significant decrease in lactate and pyruvate levels by day 7, while acetyl-CoA levels significantly increased. Citrate levels showed a mild, nonsignificant decrease. The L/P ratio significantly increased, indicating a metabolic shift from glycolysis to oxidative metabolism during early neurogenesis. Conclusions: This study demonstrates that early neuronal differentiation of WJ-MSCs involves dynamic metabolic reprogramming characterized by reduced glycolytic activity and increased oxidative metabolism. The observed shifts in lactate, pyruvate, citrate, and acetyl-CoA levels highlight the critical role of metabolic plasticity in supporting neurogenesis. These findings may inform the development of optimized differentiation protocols and enhance the therapeutic potential of stem cell-based neuroregenerative approaches.
atay et al. (Wed,) studied this question.
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