A five-step protocol for differentiation of iPS cells to pancreatic stem cells and hormone-expressing cells. A. Schematic representation of the differentiation procedure and protein expression of key markers of pancreatic differentiation. Based on D’Amour’s 5-step protocol
, this differentiation protocol is subdivided into 5 stages, and the growth factors, medium, and range of duration of each stage are shown. Several markers characteristic of each cell population are listed. Pancreatic stem cells were established after induction of stages 1 and 2. Abbreviations: CYC, KAAD-cyclopamine; RA, all-trans-retinoic acid; DAPT, γ-secretase inhibitor; Ex4, exendin-4; iPS, induced pluripotent stem cell; ME, mesendoderm; DE, definitive endoderm; PG, primitive gut tube; PF, posterior foregut endoderm; PE, pancreatic endoderm and endocrine precursor; EN, hormone-expressing endocrine cells. B. Morphology of 52 clones after induction of stages 1 and 2 (passage 2). Scale bars = 200 μm.