A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells

Abstract

Insulin-producing and -secreting cells derived from mouse pluripotent stem cells (PSCs) are useful for pancreatic development research and evaluating drugs that may induce insulin secretion. Previously, we have established a differentiation protocol to derive insulin-secreting cells from mouse embryonic stem cells (ESCs) using a combination of growth factors, recombinant proteins, and a culture substratum with net-like fibers. However, it has not been tested which materials and diameters of these fibers are more effective for the differentiation. Therefore, the present study aimed to produce net-like culture substratum formed from polyamide (PA) and polyacrylonitrile (PAN) fibers. Substrata were delineated into PA100, 300, 600, PAN100, 300, and 600 groups based on fiber diameters. The differentiation efficiencies of mouse ESCs cultured on the substrata were then examined by insulin 1 (Ins1) expression. Expression was found to be highest in PA300 differentiated cells, indicating the potential to produce high levels of insulin. To understand any differences in substratum properties, the adsorption capacities of laminin were measured, revealing that PA300 had the highest for it. We next examined the stage of differentiation affected by incubation with PA300. This showed that Sox17- and Pdx1-GFP-positive cells increased during the first step of differentiation. To show the production of insulin without absorption from the medium, we confirmed the expression of insulin C-peptide after differentiation. Finally, we tested the effects of PA300 on the differentiation of human-induced PSC, and found more Sox17-positive cells with the PA300 substratum at the definitive endoderm stage. Furthermore, these cells expressed insulin C-peptide and had glucose-responsive C-peptide secretion. In summary, our study identified and validated a novel substratum which is suitable for pancreatic differentiation of mouse and human PSCs.