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

Abstract

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