Controlling spheroid attachment improves pancreatic beta cell differentiation from human iPS cells.

Abstract

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.