The skin is a highly complex organ, responsible for sensation, protection against the environment (pollutants, foreign proteins, infection) and thereby linked to the immune and sensory systems in the neuro‐immuno‐cutaneous (NIC) system. Cutaneous innervation is a key part of the peripheral nervous system; therefore, the skin should be considered a sensory organ and an important part of the central nervous system, an ‘active interface’ and the first connection of the body to the outside world. Peripheral nerves are a complex class of neurons within these systems, subsets of functions are conducted, including mechanoreception, nociception and thermoception. Epidermal and dermal cells produce signalling factors (such as cytokines or growth factors), neurites influence skin cells (such as via neuropeptides), and peripheral nerves have a role in both early and late stages of the inflammatory response. One way this is achieved, specifically in the cutaneous system, is through neuropeptide release and signalling, especially via substance P (SP), neuropeptide Y (NPY) and nerve growth factor (NGF). Cutaneous, neuronal and immune cells play a central role in many conditions, including psoriasis, atopic dermatitis, vitiligo, UV‐induced immunosuppression, herpes and lymphomas. Therefore, it is critical to understand the connections and interplay between the peripheral nervous system and the skin and immune systems, the NIC system. Relevant in vitro tissue models based on human skin equivalents can be used to gain insight and to address impact across research and clinical needs.

There is interest in the neurological and immune components in skin, but most studies only focus on one system at a time, not both. [1,4,16] The in vitro HSE system described herein is a multilayered, full thickness skin model, containing only primary human cells, including human induced neural stem cells (hiNSCs), and tissue-inherent immune cells from lipoaspirate utilized in the hypodermis. [6] In order to compare the effect of the tissue components (i.e., hiNSCs, hypodermis, and the dermis/ epidermis), a parametric study was performed of the system in vitro. Through RNA Sequencing (RNASeq) and protein analysis it was determined that each sample group (dermis/epidermis alone, dermis/epidermis + hiNSCs, etc.) was distinct in terms of functions, gene expression profiles, and enriched biological pathways. Each sample group expressed genes related to several phenomena key to skin biology, including skin development, neuronal system development, inflammation, and immune system process.Through RNASeq, numerous genes were up-or downregulated in a distinct manner with respect to the specific HSE study group (i.e., each HSE group had distinct gene expression) that would not have been possible to identify as quickly or thoroughly by other techniques such as real-time reverse transcriptase polymerase chain reaction (qRT-PCR) or microarrays due to lower sensitivity and throughput. [17,18] Further, RNASeq allowed the identification of genes which may be important for development of an in vitro HSE model, and which were different with the study groups that contained neural or immune components. In comparison to the RNASeq results from less complex skin models (such as a keratinocyte model), the tissue models described herein identified more genes, likely due to the addition of important cell types of the skin including adipose, neural, and immune components. We speculate that such additional complexity and readouts could be useful for studies related to in vitro studies, such as insight into diseases like psoriasis. [19] The skin is a highly organized and complex organ that maintains homeostasis. The neuro-immuno-cutaneous system refers to one of the interconnected systems of the skin, in which neural, immune, and dermal cells, including neuropeptides and cytokines, are in bidirectional communication with A variety of human skin equivalents (HSEs) has been designed for clinical use or for exploratory skin research. In vitro HSE models have been used to target relationships between the skin and nervous or immune systems but have not yet considered the neuro-immuno-cutaneous (NIC) system. In this study, HSEs are described, with and without neural and immune components, to discern these types of effects. These systems are composed of only primary human cells and contain an epidermis, dermis, hypodermis (with immune cells), and human induced neural stem cells for the neuronal component. RNA sequencing is utilized to confirm differences between sample groups and to identify unique or important genes with respect to sample type. O...

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The importance of human cell-based in vitro tools to drug development that are robust, accurate, and predictive cannot be understated. There has been significant effort in recent years to develop such platforms, with increased interest in 3D models that can recapitulate key aspects of biology that 2D models might not be able to deliver. We describe the development of a 3D human cell-based in vitro assay for the investigation of nephrotoxicity, using RPTEC-TERT1 cells. These RPTEC-TERT1 proximal tubule organoids ‘tubuloids’ demonstrate marked differences in physiologically relevant morphology compared to 2D monolayer cells, increased sensitivity to nephrotoxins observable via secreted protein, and with a higher degree of similarity to native human kidney tissue. Finally, tubuloids incubated with nephrotoxins demonstrate altered Na+/K+-ATPase signal intensity, a potential avenue for a high-throughput, translatable nephrotoxicity assay.

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