Human embryonic epidermis contains a diverse Langerhans cell precursor pool
Christopher SchusterMichael MildnerMario MairhoferWolfgang BauerChristian FialaMarion PriorWolfgang EppelAndrea KolbusErwin TschachlerGeorg StinglAdelheid Elbe‐Bürger
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Abstract:
Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207- cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14+CD16- blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14- haematopoietic precursors possess a much higher differentiation potential than CD14+ precursor cells.Keywords:
Epidermis (zoology)
Skin equivalent
Cord blood
Human skin
Precursor cell
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Human skin
Epidermis (zoology)
Skin Aging
Artificial skin
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Abstract Human skin equivalents (HSEs) are three‐dimensional skin organ culture models raised in vitro. This review gives an overview of common techniques for setting up HSEs. The HSE consists of an artificial dermis and epidermis. 3T3‐J2 murine fibroblasts, purchased human fibroblasts or freshly isolated and cultured fibroblasts, together with other components, for example, collagen type I, are used to build the scaffold. Freshly isolated and cultured keratinocytes are seeded on top. It is possible to add other cell types, for example, melanocytes, to the HSE—depending on the research question. After several days and further steps, the 3D skin can be harvested. Additionally, we show possible markers and techniques for evaluation of artificial skin. Furthermore, we provide a comparison of HSEs to human skin organ culture, a model which employs human donor skin. We outline advantages and limitations of both models and discuss future perspectives in using HSEs.
Human skin
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Epidermis (zoology)
Equivalent
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Using an organotypic model of human skin, living skin equivalent (LSE) and its homogenate, monolayer cell culture, and human skin, we have studied the simultaneous transport and metabolic fate of two compounds. The LSE was maintained in an assay culture medium. When the model compounds were applied to LSE at dosages of 9.0 ± 1.2 µg/cm2, the transport of salicylic acid through human skin was 0.12 ±0.1µg/cm2/hr. Salicylic acid flux was 5.6-fold greater in LSE than in human skin. Shorter lag time of absorption was observed in LSE (∼ 1.5 hr) than in human skin (7–9 hr). Percutaneous transport across LSE was accompanied by metabolism of the compounds and there were quantitative and qualitative similarities between the metabolites produced by the LSE and human skin. When compounds were added to homogenates (LSE or skin) and to human cell cultures, the activities of the LSE and human fractions were similar. Data from the present study demonstrate that although LSE is more permeable than human skin, the artificial skin is a potentially valuable model for skin pharmacologic studies.
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As a major extracellular matrix component within the skin, collagen has been widely used to engineer human skin tissues. However, most collagen is extracted from animals. Here, we introduced recombinant human type III collagen (rhCol3) as a bioactive component to formulate bioinks for the bioprinting of a full-thickness human skin equivalent. Human dermal fibroblasts were encapsulated in the gelatin methacryloyl-rhCol3 composite bioinks and printed on a transwell to form the dermis layer, on which human epidermal keratinocytes were seeded to perform an air-liquid interface culture for 6 weeks. After optimizing the bioink formulation and bioprinting process, we investigated the effect of rhCol3 on skin tissue formation. The results suggest that a higher concentration of rhCol3 would enhance the growth of both cells, resulting in a more confluent (~100%) spreading of the epidermal keratinocytes at an early stage (3 days), compared to the rhCol3-free counterpart. Moreover, in an in vivo experiment, adding rhCol3 in the hydrogel formulation would contribute to the skin wound healing process. Taken together, we conclude that rhCol3 could act as a functional bioink component to promote basic skin cellular processes for skin tissue engineering.
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Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions and reducing the use of animals. Critical to the use of such technology is the ability to develop tissue models that accurately recreate the microanatomy of the native tissue. In this thesis, we have bioengineered novel models of neonatal and ageing human skin, that recreate the structure of human skin in vitro. These skin equivalents have been applied to investigate the mechanisms of skin ageing, such as the influence of epidermal-dermal interactions.
We have successfully bioengineered a neonatal full thickness skin equivalent that accurately recreates aspects of the microanatomy of human skin. The skin equivalent was generated using a bottom-up tissue engineering approach, whereby the human fibroblasts secrete their own endogenous extracellular matrix proteins within the porous Alvetex® Scaffold. In-depth morphological analyses demonstrate close similarities with native human skin, such as an
organised, stratified and keratinised epidermis, the presence of a robust basement membrane and the deposition of extracellular matrix proteins within the dermal compartment.
Many dermal matrices used in skin tissue engineering are not suitable for ageing studies as they cannot be tailored to recapitulate the age-related decline in extracellular matrix in vitro. We have found that incorporating ageing dermal fibroblasts within the Alvetex® Scaffold, to form a dermal equivalent, enables age-related changes, such as decreased proliferation and reduced synthesis of extracellular matrix proteins, to be recreated in vitro. These ageing dermal equivalents have been applied to investigate the influence of epidermal-dermal interactions during ageing.
Dermal interactions are thought to influence the epidermal morphology during embryogenesis and in adult skin, however there is a paucity of information regarding the importance of epidermal-dermal interactions during ageing. Tissue engineering approaches described within this thesis suggest that an ageing dermis contributes to the age-related epidermal phenotype and influences
epidermal thickness, keratinocyte differentiation and the structure of basal keratinocytes.
In the current ageing demographic, there is a requirement for an ageing skin model for academic and industrial applications. We have bioengineered novel and advanced full thickness skin equivalents representative of female ageing skin, which recreate the structure of human skin in vitro, with regards to epidermal differentiation, the presence of a basement membrane and synthesis of extracellular matrix proteins. The ageing skin equivalents also successfully
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