(D-136) Mechanical microenvironment modulates pancreatic endocrine lineage commitment

Diabetes is a commonly diagnosed disease associated with the minimal functionality or loss of pancreatic beta cells. Thus, strategies to enhance beta cell function is crucial for the development of beta cell replacement strategies. During pancreas development, beta cells are exposed to a dynamic microenvironment encompassing distinct cell-to-cell and cell-to-extracellular matrix (ECM) interactions. The ECM determines tissue morphology, which directs the transmission of cell signaling pathways translating to direct transcriptional events to coordinate lineage decisions. To study the effects of cellular interactions for promotion of functional beta cell differentiation, we developed a mechanically tunable scaffold system (MTSS) to investigate the effects of dynamic stiffness changes on endocrine cell development. The MTSS was used to culture embryonic day (E)12.5 pancreas explants. After 48 hours of culture, the pancreas explants were analyzed using quantitative PCR and RNA sequencing.

The mechanically tunable scaffold system (MTSS) is a novel device used to dynamically alter the microenvironment stiffness. The MTSS system utilizes an electrospun polycaprolactone (PCL) nanofibrous scaffold. A plunger applies tension to the fibers thereby modulating stiffness of the microenvironment on demand. On embryonic day 12.5, mouse pancreas explants were harvested and cultured on the scaffold at a low or high stiffness for 24 hours to allow for attachment. Following the initial 24 hours, the scaffold stiffness was incrementally increased or decreased over a 24-hour period to determine the effects of dynamic stiffness changes on pancreas development. Explants cultured on a static low or high stiffness were compared to those with dynamic changes in microenvironment stiffness. The cells were then lysed and RNA was purified for subsequent real time polymerase chain reaction (rtPCR) and RNA sequencing (RNAseq) analysis to assess gene expression changes.

Pancreas explants cultured on the MTSS showed a significant difference in expression of pro-endocrine marker Neurogenin3 (Ngn3), as well as other genetic markers related to pancreatic organogenesis. Culturing pancreas tissue in a low stiffness microenvironment significantly increased the expression of Ngn3, while a high stiffness decreased Ngn3 expression. This is likely due to the soft scaffolding decreasing the spreading/branching of tissue to alter cell-ECM signal transduction. Interestingly, Ngn3 was also found to have an inverse relation with expression of Notch signal pathway marker Hes1, a known Ngn3 repressor. To confirm the rtPCR results, RNAseq data demonstrated that genes associated with pancreas beta cells were significantly downregulated in explants cultured on high stiffness scaffolds. Collectively, the data suggest that the mechanical microenvironment directly influences endocrine lineage commitment. Further understanding of the physical microenvironment requirements during normal pancreas development can be used to improve the differentiation process to achieve beta cell functionality for beta cell replacement therapies.