Skin is the largest human organ and performs various functions such as protection, temperature regulation, water retention, sensation and immune defense. An organoid is a collection of organ- specific cells that develops from stem cells or organ progenitors and is capable of recapitulating specific functions of the organ. Epidermal organoids are organoids grown from keratinocytes isolated from the epidermis. Here, we describe a well-established and reproducible method for culturing human epidermal organoids (HEOs) in 7 days. We tested different media conditions to develop defined HEO growing and expansion conditions. HEOs were successfully generated from primary keratinocytes isolated from human skin as well as established keratinocyte cell lines (NKc21 and KERTr cells). We characterized these organoids to show that they resemble the human epidermis. We demonstrated that the HEOs express epidermal genes including collagen 17 (col17), keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), filaggrin (FLG), transglutaminase 1 (TGM1), and transglutaminase 3 (TGM3). We showed that as the organoids progressed from stem like basal cells to mature organoids. They lost their stemness as indicated by the downregulation of lgr5 and lgr6. They retained their basal layer markers including krt5,14,15 and col17 and the markers for the suprabasal layers, namely tgm1, krt1, krt10, tgm3, and flg were upregulated indicating the differentiation of keratinocytes. This differentiation process is hard to control in regular 2D keratinocyte culture. Overall, our data show that we were able to successfully generate HEOs that recapitulate the complexity and architecture of a human epidermis. HEOs can be derived from patients with different disorders, allowing us to generate in vitro models of skin diseases. This makes HEOs an essential tool for physiopathology research, drug development, and personalized medicine of epidermal disorders.

ESDR131 - Human epidermal organoids: establishing a reproducible stratified human epidermal organoid culture system

50th Annual Meeting of the European Society for Dermatological Research. VIRTUAL MEETING