Extended Data Figure 1 |. Overview of skin organoid induction in relation to normal human skin developmental events and its morphological changes under different treatment regimes.

a, Schematic overview of the skin organoid protocol; i) pre-aggregate hPSCs to form an aggregate, ii) induce differentiation of aggregate to form surface ectoderm and cranial neural crest cells (CNCC) by modulating TGF and BMP signaling pathways, and iii) provide sufficient environment for aggregate to mature itself into a complex skin organoid unit composed of fully stratified skin with dermal layer, producing hair follicles, sensory neurons, and cartilages. The cystic skin organoid can be transplanted and integrated into the back skin of a mouse as a planar layer and out-grow hair follicles. b, Comparison of in vitro and in vivo skin development timelines. Note that developmental timing is approximate. c, Representative differential interference contrast (DIC) and endogenous GFP fluorescence images of WA25 and DSP-GFP skin organoids on different days of differentiation under different treatment regimes. (upper) WA25 skin organoid were differentiated in E6-based medium culture under three different day-3 treatment conditions - no LDN nor FGF, LDN only, and LDN/FGF - and monitored during days 0–60. Day-3 no LDN nor FGF treated aggregates maintained cystic organoids for about 30 days of differentiation, but the organoids lost their morphology and shrunk afterwards. Day-3 LDN only treatment promoted cystic morphology, but was not sufficient to produce hair-bearing skin organoids in a consistent manner. Co-treatment of LDN and FGF on day-3 (final optimized treatment condition) was optimal for epithelial stratification and sufficient dermal layer development. Tail-like region containing mesenchymal and neuronal cells on one pole of skin organoids are visible by day-18 of differentiation. (lower) DSP-GFP skin organoids were also differentiated in E6-based medium with day-3 treatment of LDN/FGF (final optimized treatment regime). GFP⁺ epithelium is visible on the surface/edge of the sphere-like organoid, and the GFP signal intensifies as the organoids differentiate and mature further. The tail portion of the organoid appears by day 22 of differentiation (GFP⁺ signal at the tail portion presented in the day 22 image is an autofluorescence). (right) WA25 skin organoid were differentiated in CDM-based medium under three different day-4 treatment conditions - no LDN nor FGF, LDN only, and LDN/FGF. By differentiation day-55, aggregates that were treated with neither LDN nor FGF eventually lost their shape and shrunk. Day-4 LDN treatment maintained cystic organoid morphology, and occasionally induced maturation of skin organoids. Co-treatment of LDN and FGF on day-4 of differentiation was suitable for skin organoids to fully mature in a relatively consistent manner. Note: Substitution of CDM- to E6-based differentiation medium improved skin organoid development by reducing the tail portion (non-skin related mesenchymal cells) and increasing the head – skin – portion. Optimization experiments were repeated at least three times independently. Scale bars, 500 μm (all panels).

Corresponds with data/concepts in Fig. 1.