Synergistic Effect of 3D Culture and a Pancreatic Tissue-Derived ECM on Efficient Differentiation of WJ-MSCs into Pancreatic Islet-like Cells

Type 1 diabetes mellitus (T1D) is a global disease, and stem cell-derived insulin-producing cells show great promise for treatment; their <i>in vitro</i> development is limited. This suggests that the <i>in vivo</i> environment, particularly the native extracellular matrix (ECM), plays a crucial role in cell maturation. To address this, our study investigated this challenge through a two-part investigation aimed at enhancing the functional maturity of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). In the first part, we optimized a differentiation protocol by selectively identifying key small molecules. We found that a combination of CHIR98014 and Latrunculin A was crucial for improving the efficiency at different stages of differentiation. Based on this, in the second part of our study, we examined a novel approach combining an ultralow attachment-based 3D culture (ULA 3D) with a pancreas-specific decellularized ECM (pdECM) derived from porcine pancreas. We aimed to determine if this combination could enhance the differentiation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into functional insulin-producing cells. The study compared the differentiation across four groups: a standard 2D culture, ULA 3D culture, a pdECM-supported 2D culture, and the combined pdECM-supported 3D culture. We found that the definitive endoderm and pancreatic progenitor marker expression was significantly higher in the ULA group, especially when supported by the pdECM. Pancreatic endocrine gene and protein expressions, including <i>PDX1</i>, <i>NGN3</i>, and <i>INS</i>, were notably enhanced in this combined 3D cultured group. Crucially, the pdECM-supported 3D culture group showed higher levels of glucose-stimulated insulin secretion, confirming the functional maturity of the differentiated cells. This study demonstrates that creating and integrating a tissue-specific pdECM into a 3D culture system provides a more physiologically relevant microenvironment, significantly improving the differentiation and function of WJ-MSCs and holding great potential for effective regenerative therapies for T1D.