Innovative Approaches to Neural Differentiation: Chondroitin 4-Sulfate and Chondroitinase Induce Differentiation in Human-induced Pluripotent Stem Cells

Background: Stem cells have recently gained prominence in regenerative medicine, particularly in the treatment of neurological disorders. As a result, Human-induced Pluripotent Stem Cells (hiPSCs) have become a significant focus. Objective: This study aimed to differentiate hiPSCs into neural lineages under in vitro conditions using forskolin and retinoic acid in an induction medium combined with chondroitin 4-sulfate and chondroitinase. Methods: Optimal component concentrations were determined using the MTT assay and acridine orange/ethidium bromide (AO/EB) staining. Subsequently, neural-specific genes (NSE, MAP-2, β-tubulin III, Oligo-2, and GFAP) and proteins (gamma enolase, MAP-2, and β-tubulin III) were assessed using Real-time PCR analysis and immunofluorescence staining to provide a comprehensive evaluation of differentiated cells. Results: Our study demonstrated a significant enhancement in neural-specific gene and protein markers during the 7th and 14th days of differentiation in the presence of combined chondroitin 4-sulfate and chondroitinase, demonstrating a higher efficacy compared with the application of isolated enzymes or substrates. Conclusion: These findings emphasize the potential importance of chondroitin 4-sulfate and chondroitinase as important factors in promoting the neural differentiation of hiPSCs. It seems that chondroitin 4-sulfate may activate cellular signaling pathways that are effective in inducing neural differentiation. Our findings in this research provide new opportunities to advance regenerative therapies for neurological disorders.