Fig 1: PSO development and optimization through morphology-based early screening: (a) overview of PSO differentiation protocol with key growth factors. (b) Identification of PSO distinct morphology two weeks into the differentiation protocol. Flow cytometry analysis of the organoid cell suspension collected at day 45 shows a correlation between ITGA6/PDGFRα expression and epidermis development. H&E shows proper development of an epidermis when dense iKCs sorted at day 45 and cultured to passage 2 are seeded on top of a primary FB dermis, and a lack of epidermis formation when cyst iKCs are used. (c) The morphology-based screening approach was used to select a narrow range of BMP-4 concentrations for optimization of RDEB PSO differentiation, and flow cytometry and functional characterization by seeding iKCs on a primary FB dermis show 4 ng/ml yields iKCs that form a thicker epidermis in vitro. Scale bars: 100 µm.