Speaker
Description
"Introduction
Adipose-derived stem cell (ASC) are increasingly recognized as a vital tool for cell-based therapy. Hypoxic pre-conditioning of ASCs provides thier functional enhancement and makes the cells more attractive for translational science. Recently, ASCs from livestock animals have gained increased attention due to their promising role in developing regenerative medicine protocols that can be utilized in veterinary medicine and humans. In searching for alternative sources of stem cells to facilitate wound healing we isolated and characterized of ASCs from fat of domestic pigs (pASCs). We examined effect of hypoxia (1% O2) on pASCs functional features, proteome and secretome, and we determined comparative studies of the effect of hypoxia- and normoxia-cultured pASCs (pASCs-Hyp and pASCs-Nor, respectively) on the healing process of full-thickness cutaneous wounds in mice.
Methodology
pASCs were isolated from the subcutaneous fat of domestic pigs and characterized by morphology, surface markers expression and multilineage differentiation. The effect of hypoxia (1% O2) on pASCs (1) proteome was examined using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry; (2) secretome was determined in pASCs conditioned media (CM) using cytometric bead array technology. Bioactivity of pASCs-CM was determined in terms of their potential to modulate the characteristics of skin cells in vitro. For the in vivo studies pASCs-Hyp and pASCs-Nor were intradermally injected into full-thickness cutaneous wounds in C57BL/6 adult mice. Wound healing process was monitored macroscopically. In skin samples collected at days 5, 14 and 21 post-wounding we examined re-epithelialization, collagen deposition, and the expression of pro-fibrotic (Tgfβ1, Wnt signaling members) and anti-scarring (Tgfβ1, hyaluronic acid) markers of healing.
Results
Immunophenotypic and functional analysis showed that pASCs meet the criteria of mesenchymal stem cells. Proteomic analysis revealed 70 differentially abundant proteins between pASCs cultured under hypoxia (1% O2) or normoxia (21% O2). Differentially expressed proteins were predominantly involved in cell metabolism, regulation of focal and intracellular communication, and attributed to wound healing. Functional examination of hypoxic pASCs demonstrated acquisition of contractile abilities in vitro. Analysis of pASCs secretome revealed that exposure to hypoxia increased levels of vascular endothelial growth factor (VEGF) in CM-Hyp compared to CM-Nor. CM-Hyp promoted the migratory ability of pig keratinocytes and delayed migration of pig dermal fibroblasts. The formation of three-dimensional endothelial cell networks was improved in the presence of CM-Hyp. In vivo studies on mouse model of wound healing demonstrated that the effect of pASCs-Hyp on cutaneous healing was superior to that of pASCs-Nor in terms of re-epithelialization, scar size, hyaluronic acid content, and the expression of Tgfβ1 and Tgfβ3. However, wounds administered with pASCs-Hyp exhibited increased expression of Wnt signaling members including Wnt10a, Wnt11, and β-catenin, which are associated with scar-forming wound repair.
Conclusions
Hypoxia pre-conditioning impacts the pASC proteome signature, secretome and functionality. In vivo translantional studies demonstrate the pASCs safety and efficacy and indicate that the xenotransplantation of hypoxia-preconditioned pASCs might be considered a therapeutically relevant approach in the wound healing context.
This work was supported by the National Science Centre, Poland, grant SONATA 13, No. 2017/26/D/NZ5/00556."
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