The ability to harvest and culture stem cell populations from various human postnatal tissues is central to regenerative medicine applications, including tissue engineering. The discovery of multipotent mesenchymal stem cells within the stromal fraction of adipose tissue prompted their use for the healing and reconstruction of many tissues. Here, we examined the influence of adipose-derived stem/stromal cells (ASCs) on skin's regenerative processes, from a tissue engineering perspective. Using a self-assembly approach, human skin substitutes were produced. They featured a stromal compartment containing human extracellular matrix endogenously produced from either dermal fibroblasts or adipose-derived stem/stromal cells differentiated or not toward the adipogenic lineage. Human keratinocytes were seeded on each stroma and cultured at the air-liquid interface to reconstruct a bilayered skin substitute. These new skin substitutes, containing an epidermis and a distinctive stroma devoid of synthetic biomaterial, displayed characteristics similar to human skin. The influence of the type of stromal compartment on epidermal morphogenesis was assessed by the evaluation of tissue histology, the expression of key protein markers of the epidermal differentiation program (keratin [K] 14, K10, transglutaminase), the expression of dermo-epidermal junction components (laminins, collagen VII), and the presence of basement membrane and hemidesmosomes. Our findings suggest that adipose-derived stem/stromal cells could usefully substitute dermal fibroblasts for skin reconstruction using the self-assembly method. Finally, by exploiting the adipogenic potential of ASCs, we also produced a more complete trilayered skin substitute consisting of the epidermis, the dermis, and the adipocyte-containing hypodermis, the skin's deepest layer.
In the past years, adipose tissue has spurred a wide interest, not only as a source of adult multipotent stem cells but also as a highly eligible tissue for reconstructive surgery procedures. Tissue engineering is one field of regenerative medicine progressing at great strides in part due to its important use of adipose-derived stem/stromal cells (ASCs). The development of diversified technologies combining ASCs with various biomaterials has lead to the reconstruction of numerous types of tissue-engineered substitutes such as bone, cartilage, and adipose tissues from rodent, porcine, or human ASCs. We have recently achieved the reconstruction of connective and adipose tissues composed entirely of cultured human ASCs and their secreted endogenous extracellular matrix components by a methodology known as the self-assembly approach of tissue engineering. The latter is based on the stimulation of ASCs to secrete and assemble matrix components in culture, leading to the production of cell sheets that can be manipulated and further assembled into thicker multilayer tissues. In this chapter, protocols to generate both reconstructed connective and adipocyte-containing tissues using the self-assembly approach are described in detail. The methods include amplification and cell banking of human ASCs, as well as culture protocols for the production of individual stromal and adipose sheets, which are the building blocks for the reconstruction of multilayered human connective and adipose tissues, respectively.
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