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Construction of Human Primary Epidermal Organoids and Modeling of Dermatophyte Infection

ABSTRACT: Despite the strong need for the large-scale generation of human epidermal cells for cell therapy of extensive and severe skin injury, tissue-engineered skin construction and dermatological investigation, the effectiveness of current culture methods is limited and have yet to meet the huge demands sufficiently. Here, we report the establishment of a simple and robust 3D culture system using chemical defined, serum-free medium supplemented with small molecules and growth factors, tailored for human epidermal organoids generation and expansion. The conditions allowed 10,000-fold expansion of the primary epidermal cells within 6 weeks, with the cell architecture and gene expression preserved. We further noticed that only Integrin α6-positive basal stem cells freshly isolated from human epidermis could form expandable epidermal organoids. The epidermal cells inside of the organoids also have the potential to reconstruct multi-layered human epidermis equivalents under classical air-liquid interface condition. Remarkably, the epidermal organoids enable to model the anthropophilic and epidermis-restricted Trichophyton Rubrum infection. This model faithfully mimics and reflects the clinical pathological reactions of reinforced physical barrier by promoting keratinocytes differentiation and accelerated formation of the stratum corneum for adaptation and resistance to infection. Furthermore, the infection model also suggested that constraining host IL-1 signaling may underline the high degree of adaptation of Trichophyton Rubrum to human skin, and the tendency to be chronic and recurrent infection. Thus, human epidermal organoids present a powerful platform for modeling human dermatology, skin tissue engineering and cell therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE134403 | GEO | 2020/03/01

REPOSITORIES: GEO

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Project description:Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To address this hypothesis a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae which causes highly inflammatory cutaneous infections in humans and rodents. Microarray analysis revealed a distinct in vivo protease gene expression profile in the fungal cells, which is surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro expressed proteases others were activated specifically during infection. These enzymes are therefore suggested to fulfill important functions that are not exclusively associated with the degradation of keratin. As the most upregulated in vivo specific A. benhamiae sequence we discovered the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation. In addition, our approach identified other candidate pathogenicity related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. This first broad transcriptional profiling approach during dermatophyte infection gives new molecular insights into pathogenicity associated mechanisms that make these microorganisms the most successful etiologic agents of superficial mycoses. Keywords: Two-condition experiment, strong proteolytic activity in the supernatant versus no proteolytic activity or infected tissue versus no proteolytic activity Three independently prepared A. benhamiae replicates grown in each of the three media, Sabouraud, soy and keratin-soy medium (designated SabA/B/C, soyA/B/C and keratin-soyA/B/C) were used. ARN from skin samples and fungus together of Guinea Pig infected with A. benhamieae were prepared. Pairwise transcriptional comparisons, i.e. soy versus Sabouraud, keratin-soy versus Sabouraud and Guinea Pig infected versus Sabouraud were done. The total number of slides in this study was 18.

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Construction of Human Primary Epidermal Organoids and Modeling of Dermatophyte Infection

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