Generation of human skin organoids from pluripotency (Admin Supplement)

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ABSTRACT Skin morphogenesis and homeostasis necessitate the assembly of diverse cells originating from different embryonic tissues, which poses a considerable engineering challenge when aiming to construct complex and easily assembled skin in vitro. Such constructs could have significant applications, including cell therapy for burn and wound treatment. The specific issue lies in the commonly used cells for in vitro skin derivation; they lack the capacity to form skin appendages, such as hair follicles and sweat glands, and accessory cells, like melanocytes and adipocytes. Therefore, identifying the pivotal cell signaling mechanisms required to differentiate appendage- bearing skin from progenitor cells, like human pluripotent stem cells (hPSCs), or to stimulate skin appendage induction from adult skin cells, represents a critical barrier to progress. While we can efficiently generate epidermal cells from hPSCs in vitro, the regulation of dermal progenitor cell induction and other components of the skin microenvironment remains uncertain. As such, our long-term objective is to ascertain the chemical and physical signals necessary to recapitulate the development of human skin progenitor cells and reconstitute complex skin. This supplementary project builds upon the progress made in our parent project, "Generation of human skin organoids from pluripotency," by introducing a new aim focused on modeling pain pathways in skin organoids. Firstly, we will test the hypothesis that nociceptive neurons emerge in skin organoids and form specialized nerve endings associated with terminal Schwann cells, akin to human fetal and newborn skin. Additionally, we aim to establish a methodology for generating stereotyped nociceptive innervation of skin organoids grown in a tissue-chip environment for accessible live-cell imaging. Secondly, we will examine the hypothesis that macrophages co-derived in skin organoids can modulate the functional activity of nociceptors, thereby mimicking cell-cell interactions implicated in pain initiation and resolution. Successful completion of these aims will provide preliminary data for innovative new imaging-based investigations into the mechanisms of acute and chronic pain and inflammation in the skin. In alignment with the parent award, we also expect to gain considerable insight into the earliest stages of human skin and pain circuit development for researchers studying skin organogenesis. Furthermore, our efforts may inform strategies for resolving pain associated with conditions that require tissue reconstruction using skin organoid-derived cells.