Optimal Protocol for Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells into Pancreatic β-like Cells.

Background: Diabetes mellitus, characterized by β-cell dysfunction and loss, results in impaired insulin secretion and chronic metabolic complications. Mesenchymal stem cells represent a promising source for β-cell regeneration owing to their endodermal differentiation potential. This study optimized the differentiation of human tonsil-derived mesenchymal stem cells (TMSCs) into pancreatic β-like cells through the comparative evaluation of two signaling-based protocols.

Methods: TMSCs were isolated from human tonsils, and passages 5-7 were used for characterization and β-like cell differentiation. Two protocols were tested in both two-dimensional (2D) and three-dimensional (3D) cultures: Protocol I induced direct differentiation into pancreatic β-like cells without sequential developmental stages, whereas Protocol II followed stepwise progression from definitive endoderm to pancreatic progenitors, and finally pancreatic β-like cells.

Results: In 2D culture, cells cultured under protocol II maintained a typical morphology and high confluency, whereas cells cultured under protocol I displayed a thinner shape with reduced confluency. Protocol II effectively induced definitive endoderm differentiation, as demonstrated by the increased expression of SOX17, FOXA2, and CXCR4 at the mRNA and protein levels. Both protocols upregulated pancreatic progenitor (HNF6 and NGN3) and pancreatic β-cell markers (Insulin, NKX6.1, and PDX1), but protocol II consistently produced higher pancreatic β-cell marker expression, except for MafA. In 3D spheroid culture, insulin, PDX1, and MafA levels increased, with protocol II showing superior expression over protocol I. Additionally, differentiated cells exhibited glucose-responsive C-peptide secretion, indicating the acquisition of early β-cell-like functional properties.

Conclusion: Stepwise, developmentally guided differentiation (Protocol II) more effectively directs TMSCs toward pancreatic β cell lineage commitment, highlighting the importance of stage specific signaling cues and supporting the potential of TMSCs for pancreatic β cell-based regenerative applications.