A Stepwise Method to Derive Functional Pancreatic Islet-like Clusters from Human Pluripotent Stem Cells

The most common treatment for people with people with type 1 diabetes (T1D) requires injections of exogenous insulin to regulate blood glucose homeostasis. A longer-term therapeutic solution for T1D has emerged whereby patients may regain insulin independence through transplantation of donated human pancreatic islets, which contain insulin-secreting beta (β)-cells. Donated islets, however, are in short supply. Much effort has been dedicated to using human pluripotent stem cell (hPSC)-derived islets (SC-islets) as an unlimited source of insulin-secreting β-cells. Here, we describe a robust and reproducible 40-day, seven-stage stepwise process for generating hPSC-derived SC-islets. Our differentiation protocol aims to mimic in vivo pancreas development using stage-specific signals to promote the generation of SC-islets. The initial maintenance and the first four stages are performed in two-dimensional monolayer culture; the subsequent three stages require three-dimensional culture in microwells to induce the formation of islet-like clusters. The SC-islets generated at the end of the procedure respond to glucose challenges, reliably express appropriate endocrine markers, contain intracellular insulin, and display an islet-like architecture. Most SC-islets also partially express maturity markers that are often challenging to activate in vitro. These protocols thus provide a scalable platform for generating SC-islets for disease modeling and cell replacement studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Thawing, recovery, and passaging of cryopreserved hPSCs Support Protocol 1: Preparation of solutions and media for hPSC maintenance and expansion Support Protocol 2: Preparation of Matrigel-coated plates for passaging hSPCs Basic Protocol 2: Differentiation of hPSCs to SC-islets.