Human-induced pluripotent stem cell- (iPSC-) derived insulin-producing cells (IPCs) can be used for islet cell transplantation into type 1 diabetic patients and as patient-specific cells for the development of novel antidiabetic drugs. However, a method is needed to generate functional IPCs from iPSCs and simplify the protocol. We compared combinations of small molecules that could induce the differentiation of cells into a definitive endoderm and preferentially into islet precursor cells. When generated using an optimal combination of small molecules, IPCs secreted insulin in response to glucose stimulation. We constructed spheroid IPCs and optimized the culture and maturation conditions. Quantitative PCR revealed that the expression of definitive endoderm-specific markers differed depending on the combination of the small molecules. The small molecule, N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine, induced the differentiation of cells into functional IPCs by inhibiting Sonic hedgehog signaling. Images of the 2D culture showed that IPCs formed spheroids from day 5 and continuously secreted insulin. We developed a simple differentiation method using small molecules that produced functional IPCs that responded to glucose stimulation within a relatively short period. We posit that this method along with further refinement of the differentiation process can be applied to culture IPCs that can be used in clinical trials.

Background: Human-induced pluripotent stem cell (iPSC)-derived insulin-producing cells (IPCs) can be used for islet cell transplantation in type 1 diabetic patients and as patient-specific cells for the development of novel anti-diabetic drugs. Therefore, it is necessary develop a method for generating functional IPCs from iPSCs and simplifying the stepwise protocol. Methods: We compared combinations of small molecules that could efficiently induce the differentiation of cells into a definitive endoderm, and preferentially into islet precursor cells. IPCs, generated using the optimal combination of small molecules, were confirmed to demonstrate insulin secretion in response to glucose stimulation. Finally, we re-constructed spheroid IPCs and verified the optimized culture and maturation conditions. Results: It was confirmed by quantitative polymerase chain reaction that definitive endoderm-specific markers were expressed differently depending on the combination of the small molecules used. Small molecule SANT-1 induced the differentiation of cells into functional IPCs by acting as an inhibitor of Sonic hedgehog signaling. Images of 2D culture showed that IPCs were spheroid-shaped from day 5 and demonstrated sustained insulin secretion. We developed a simple differentiation method using small molecules that produced functional IPCs that responded efficiently to glucose stimulation in a relatively short time. Conclusions: We posit that this method along with a method that refines the process of differentiation can be used for growing IPCs that can be employed in clinical trials.