Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro.

Chronic wounds, such as leg ulcers associated with sickle cell disease, occur as a consequence of a prolonged inflammatory phase during the healing process. They are extremely hard to heal and persist as a significant health care problem due to the absence of effective treatment and the uprising number of patients. Indeed, there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Development in skin engineering leads to a small catalogue of available substitutes manufactured in Good Manufacturing Practices compliant (GMPc) conditions. Those substitutes are produced using primary cells that could limit their use due to restricted sourcing. Here, we propose GMPc protocols to produce functional populations of keratinocytes and fibroblasts derived from pluripotent stem cells to reconstruct the associated dermo-epidermal substitute with plasma-based fibrin matrix. In addition, this manufactured composite skin is biologically active and enhances in vitro wounding of keratinocytes. The proposed composite skin opens new perspectives for skin replacement using allogeneic substitute.

Behaviour of endothelial cells in a tridimensional in vitro environment.

Angiogenesis is a fundamental process in healing, tumor growth, and a variety of medical conditions. For this reason, in vitro angiogenesis is an area of interest for researchers. Additionally, in vitro angiogenesis is important for the survival of prevascularized tissue-engineering models. The aim of this study was to observe the self-tubular organization behaviour of endothelial cells in the self-assembly method. In this study, bilayered and dermal substitutes were prepared using the self-assembly method. Histological, immunostaining, and biochemical tests were performed. The behavioural dynamics of endothelial cells in this biological environment of supportive cells were observed, as were the steps of the in vitro angiogenic cascade with self-organizing capillary-like structures formation. The epidermal component of the substitutes was seen to promote network expansion and density. It also increased the quantity of angiogenic factors (VEGF and Ang-1) without increasing the proinflammatory factor (IL-8). In addition, the increased MMP activity contributed to matrix degradation, which facilitated capillary formation.

Study of in vitro capillary-like structures in psoriatic skin substitutes.

Angiogenesis is one of the important hallmarks of psoriasis. The extension of the superficial microvascular structure and activated pro-angiogenic mediators in psoriasis seem to be important factors involved in the pathology. According to the changes of superficial microvasculature in psoriatic lesions, anti-angiogenic treatment could be a promising therapeutic strategy for psoriasis. The aim of this study was to construct an in vitro vascularized psoriatic skin substitute for fundamental research. Psoriatic fibroblasts and keratinocytes were isolated from psoriatic plaque biopsies, while healthy fibroblasts and keratinocytes, as well as microvascular endothelial cells, were isolated from healthy skin biopsies of cosmetic breast surgery. Psoriatic and healthy skin substitutes with and without endothelial cells were produced using the self-assembly approach. Afterward the substitutes were examined by histology, immunofluorescence studies, and three-dimensional (3D) confocal microscopy. Histological analysis and immunofluorescence staining of specific markers for endothelial cells (von Willebrand, PECAM-1 [CD31], and VE-cadherin [CD144]) and basement membrane component (collagen IV) demonstrated that endothelial cells have the ability to form capillary-like tubes. Moreover, the 3D branched structure of the capillary-like structures and an eagle eye view of them were observed by confocal microscopy. Also the semiquantification of capillary-like tubes (CLTs) was carried out with a 3D eagle eye view of substitutes, and more CLTs were observed in psoriatic substitutes. These results suggest that it is possible to observe 3D capillary-like structures in the self-assembled psoriatic skin substitutes, which could become a good in vitro testing model for anti-angiogenic drug research, and facilitate the study of this complex pathology, which links angiogenesis to its development.