Gene expression profile at single cell level of newborn and adult mouse dorsal skin organoids

The formation of tissue patterns is critical for organ functions in development and regeneration. To advance the use of organoids for organ regeneration, we learn principles that govern the skin organoids to regenerate hairs. We show the formation of epidermal-dermal coupled cysts function as competent morphogenetic units (CMU) which harbor the ability of skin organoids to regenerate. ScRNA-sequencing shows the emergence of cell types and new cell interactions during CMU formation. In newborn skin organoids, epidermal cells undergo apical-basal polarization via the IFNr-PKR-PKC signaling module. Dermal-Tgfb regulates Gsk3 to establish basement membranes between the epidermal cyst and dermal cells. Meanwhile, VEGF signaling mediates dermal cell attachment to the cyst. Adult cells cannot form organoids but can be induced to generate CMUs and regenerate hairs by adding IFNr or VEGF. We compare the similar principles and different paths used to establish morphogenetic competency in developing skin, wound-induced hair neogenesis, and organoids.

Cells were derived from the dorsal skin of neonatal mice within 24 hours of birth. The skin was floated in 0.25% trypsin solution at 4°C to separate into the dermis and epidermis. Epidermal cells were cut with scissors, pipetted, and filtered through a 70um cell strainer followed by centrifugation. The dermis was digested in 0.35% collagenase for 20 minutes, then filtered through a 70um cell strainer followed by centrifugation. The dissociated epidermal cells and dermal cells were mixed at a ratio of 1:9 and were dropped onto the upper chamber of a transwell culture insert, and the lower chamber was filled with 700ul DMEM/F12 (Corning) culture medium containing 10% FBS (Gibco). For adult cell culture, the growth factors were added from D0 to D4, followed by adding MMP14 recombinant protein from D4 to D7. PKC inhibitors were added throughout the cultivation period. The cells were cultured in a 5% CO2 at 37°C incubator, with the culture medium being changed every other day.