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. 2022 Sep 16;25(10):105150. doi: 10.1016/j.isci.2022.105150

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© 2022 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Generation of air-liquid interface (ALI)-skin organoids

(A) Schematic overview and timeline of the culture protocol for generating ALI-skin organoids from hiPSCs. Day 0 refers to when hiPSC colonies were detached to form EBs and an overview of the ALI-skin organoid culture model.

(B) Bright-field images of ALI-skin organoids after 0, 2, 4, and 6 days of culture in dry conditions. Scale bars, 50 μm, and H&E staining of ALI-skin organoids after 0, 2, 4, and 6 days of culture in dry conditions. Scale bars, 100 μm.

(C) Immunostaining for epidermal barrier markers in ALI-skin organoids after 0, 2, 4, and 6 days of culture in dry conditions and in human adult skin. Cornified (Filaggrin; green) and basal (KRT5; red) epidermal layer markers are shown on the top, and spinous (KRT10; green) and granular (Loricrin; red) epidermal layer protein markers are characterized on the bottom panel. Scale bars, 50 μm.

(D) Immunostaining for dermal layer markers (Vimentin and Collagen 3; green) and basal epithelial layer markers (KRT5; red) in ALI-skin organoids after 6 days in dry conditions (top panel) and in human adult skin (bottom panel). Scale bars, 50 μm.

(E–J) Hair follicle characterization in ALI-skin organoids. Shown are immunostaining images for Sox2⁺ dermal papilla (arrows; left and right) (E), melanocytes (melanA) found in elongated hair follicles (arrow; left) and epidermal layer (arrow; right) (F), KRT15⁺KRT5⁺ bulge stem cells (arrow; left) and NFATc1⁺KRT5⁺ bulge stem cells (arrow; right) (G), KRT17⁺KRT5⁺ outer sheaths of hair shafts; dashed box, magnified region (arrow; right) (H), KRT71⁺ inner sheaths of hair shafts (arrowhead; left), AE13⁺ hair shaft cortex (asterisk; left), and ⍺SMA⁺KRT5⁺ dermal sheath (arrow; right) (I), and ki67⁺p63⁺ hair matrix cells, dashed box, magnified region (arrow; right) (J). Scale bars, 50 μm.