Abstract
Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.
Highlights
Cell-based engineered skin substitutes are promising to treat difficult-to-heal acute and chronic wounds such as large/deep burns, ulcers resistant to conventional therapies or surgical wounds [1,2,3,4,5]
All cells showed a fibroblast-like morphology (Figure 1A, upper left panel). This was supported by immunofluorescence data showing that after triple staining of P4H, K14 and nuclei (Figure 1A, upper middle panel), the fetal fibroblasts expressed P4H but not K14 and confirmed by flow cytometry analysis demonstrating that 99.2% of cells expressed the prolyl-4-hydroxylase, a marker of fibroblasts (Figure 1A, upper right panel)
Since the 70 s, increasing attention is given to fetal wound healing with the observations that fetal skin heals rapidly without scar formation [18]
Summary
Cell-based engineered skin substitutes are promising to treat difficult-to-heal acute and chronic wounds such as large/deep burns, ulcers resistant to conventional therapies or surgical wounds [1,2,3,4,5]. Cultured autologous epidermal cell-based therapy is used for more than two decades as permanent wound coverage for large burns [6]. Allogeneic cell-based engineered skin substitutes have been developed Where they offer off-the-shelf temporary wound coverage acting as biologically active dressings releasing growth factors, cytokines and extra cellular matrix (ECM) components essential for proper wound healing, they are susceptible of immune rejection [7,8]. Among these skin substitutes, bilayered constructs associating neonatal foreskin epidermal and dermal cell layers are the most developed
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