Microporous scaffolds support assembly and differentiation of pancreatic progenitors into ß-cell clusters.

Human pluripotent stem cells (hPSCs) represent a promising cell source for the development of ß-cells for use in therapies for type 1 diabetes. Current culture approaches provide signals to mimic a temporal control of organogenesis to drive the differentiation towards ß-cells. However, spatial control may represent an opportunity to improve the efficiency and manufacturing of ß-cells. Herein, we adapted the current culture systems to microporous biomaterials with the hypothesis that the pores can guide the assembly of pancreatic progenitors into clusters of defined size that can influence maturation. The scaffold culture allowed hPSC-derived pancreatic progenitors to form clusters at a consistent size as cells differentiated. By modulating the scaffold pore sizes, we observed 250-425 µm pore size scaffold cultures augmented insulin expression and key ß-cell maturation markers compared to cells cultured in suspension. Furthermore, when compared to suspension cultures, the scaffold culture showed increased insulin secretion in response to glucose stimulus indicating the development of functional ß-cells. In addition, scaffolds facilitated cell-cell interactions enabled by the scaffold design and supported cell-mediated matrix deposition of extracellular matrix (ECM) proteins associated with the basement membrane of islet cells. We further investigated the influence of ECM on cell development by incorporating an ECM matrix on the scaffold prior to cell seeding; however, their presence did not further enhance maturation. These results suggest the microporous scaffold culture provides a conducive environment that drives in vitro differentiation of hPSC-derived insulin-producing glucose-responsive ß-cells and demonstrates the feasibility of these scaffolds as a biomanufacturing platform. STATEMENT OF SIGNIFICANCE: Cell therapy for diabetes is a promising strategy, yet generating limitless insulin-producing mature ß-cells from human pluripotent stem cells (hPSCs) remains a challenge. Current hPSC differentiation methods involve media containing signals to drive maturation toward ß-cells and spontaneous cluster formation. Herein, we sought to provide spatial cues to guide assembly of cells into 3D structures by culture within the pores of a microporous scaffold. The scaffolds direct cell-cell interactions within the pores and provide a support for cell-mediated matrix deposition that collectively creates a niche to promote functional hPSC-derived ß-cell clusters. These scaffolds for 3D culture may contribute to hPSC differentiation methods for the generation of ß-cells that can treat patients with diabetes.