Abstract
Human induced pluripotent stem cells (hiPSCs) are considered a promising source of pancreatic β-cells for the treatment of diabetes. However, this approach is limited by issues such as low efficiency and high cost. Here, we have developed a new protocol to induce insulin-producing cells. To reduce costs, we decreased the number of reagents and replaced protein reagents with chemical compounds. In this method, we increased induction efficiency with ascorbic acid (vitamin C) and an ALK5 inhibitor, RepSox. In 2D culture, the majority of cells were immature β-cells with low glucose-stimulated insulin secretion. Transferring to 3D culture immediately after endocrine progenitor cell differentiation, however, improved glucose-stimulated insulin secretion. This simplified method will contribute to realizing transplantation therapy of β-cells using iPSCs.
Highlights
Type 1 diabetes results from the destruction of pancreatic β-cells by an autoimmune process resulting in patients having little endogenous control of their blood glucose levels
Freeze-stored Human induced pluripotent stem cells (hiPSCs) were thawed and cultured on 6-cm dishes coated with Matrigel (Corning) in serum-free mTeSR1 or mTeSR Plus medium (STEMCELL Technologies), without mouse embryonic fibroblast feeder cells, at 37 ̊C under 5% CO2 in air. hiPSCs were cultured with mTeSR1 or mTeSR Plus medium with 10 μM Rho-associated kinase inhibitor (Y-27632; Adooq Bioscience) and passaged at 1:20–1:50 when hiPSCs reached about 80% confluency using 0.02% EDTA-PBS
At stage 1, Ac and CHIR were added to the medium to differentiate the human iPSC line, 201B7 cells, into the definitive endoderm
Summary
Type 1 diabetes results from the destruction of pancreatic β-cells by an autoimmune process resulting in patients having little endogenous control of their blood glucose levels. Even with insulin injections, blood glucose levels are difficult to properly control, and patients are under high stress due to the lifelong need for injections. One of the best options to overcome these issues is transplantation of pancreatic islet cells, including β-cells. This approach is limited by an insufficient number of donors. Many studies have recently been using iPSCs rather than ES cells [3,4,5,6], as using iPSCs from the patient negates the issue of both limited donors and transplant rejection
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