We detail a six-stage planar differentiation methodology for generating human pluripotent stem cell–derived pancreatic β cells (SC-β cells) that secrete high amounts of insulin in response to glucose stimulation. This protocol first induces definitive endoderm by treatment with Activin A and CHIR99021, then generates PDX1+/NKX6-1+ pancreatic progenitors through the timed application of keratinocyte growth factor, SANT1, TPPB, LDN193189 and retinoic acid. Endocrine induction and subsequent SC-β-cell specification is achieved with a cocktail consisting of the cytoskeletal depolymerizing compound latrunculin A combined with XXI, T3, ALK5 inhibitor II, SANT1 and retinoic acid. The resulting SC-β cells and other endocrine cell types can then be aggregated into islet-like clusters for analysis and transplantation. This differentiation methodology takes ~34 d to generate functional SC-β cells, plus an additional 1–2 weeks for initial stem cell expansion and final cell assessment. This protocol builds upon a large body of previous work for generating β-like cells. In this iteration, we have eliminated the need for 3D culture during endocrine induction, allowing for the generation of highly functional SC-β cells to be done entirely on tissue culture polystyrene. This change simplifies the differentiation methodology, requiring only basic stem cell culture experience as well as familiarity with assessment techniques common in biology laboratories. In addition to expanding protocol accessibility and simplifying SC-β-cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-β cells from a wide variety of cell lines from various sources, broadening its applicability.

我们详细介绍了一种六阶段平面分化方法，用于生成人类多能干细胞衍生的胰腺 β 细胞（SC-β 细胞），该细胞在葡萄糖刺激下分泌大量胰岛素。该协议首先通过激活素 A 和 CHIR99021 处理诱导定形内胚层，然后通过定时应用角质形成细胞生长因子、SANT1、TPPB、LDN193189 和视黄酸生成 PDX1+/NKX6-1+ 胰腺祖细胞。内分泌诱导和随后的 SC-β 细胞规范是通过由细胞骨架解聚化合物 latrunculin A 与 XXI、T3、ALK5 抑制剂 II、SANT1 和视黄酸组成的混合物实现的。然后可以将产生的 SC-β 细胞和其他内分泌细胞类型聚集成胰岛样簇，用于分析和移植。这种分化方法需要大约 34 天的时间来生成功能性 SC-β 细胞，另外还需要 1-2 周的时间来进行初始干细胞扩增和最终细胞评估。该协议建立在大量先前用于生成 β 样细胞的工作的基础上。在本次迭代中，我们消除了在内分泌诱导过程中对 3D 培养的需求，从而可以完全在组织培养聚苯乙烯上生成高功能 SC-β 细胞。这种变化简化了分化方法，只需要基本的干细胞培养经验以及熟悉生物学实验室中常见的评估技术。除了扩展协议可访问性和简化 SC-β-cell 生成之外，