5-Aza-2'-Deoxycytidine (5-Aza-dC, Decitabine) Inhibits Collagen Type I and III Expression in TGF-ß1-Treated Equine Endometrial Fibroblasts.

Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-ß1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-ß1-treated mare endometrial fibroblasts and the use of 5-aza-2'-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-ß1 and of 5-aza-dC on DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), COL1A1, COL3A1, and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-ß1 upregulated DNMT3A transcripts and collagen secretion. In TGF-ß1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-ß1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis.

Comparative studies on the effect of pig adipose-derived stem cells (pASCs) preconditioned with hypoxia or normoxia on skin wound healing in mice.

Adipose-derived stem cells (ASCs) from human and animal fat have emerged as therapeutic alternatives for damaged tissues. Pre-conditioning of ASCs with hypoxia results in their functional enhancement, which might facilitate the process of healing. However, there is still a critical need for large-scale preclinical studies to reinforce the translation of these findings into clinical practice for humans and in veterinary medicine. Here, we adapted a full-thickness excisional skin wound mouse model to evaluate and compare the effect of pig adipose-derived stem cells (pASCs) cultured under normoxia (pASCs-Nor) or hypoxia (pASCs-Hyp) on the healing process. We show that pASCs-Hyp accelerated re-epithelialization, increased hyaluronic acid (HA) content, and decreased scar elevation index (SEI) during the late stage of healing (day 21). Transplantation of pASCs-Hyp also promoted expression of angiogenic marker VegfA and decreased levels of pro-scarring Tgfß1. Mice tolerated xenotransplantation of the pASCs with no impact on macrophage (CD68 -positive cell) content. However, wounds treated with pASCs-Hyp exhibited decreased elasticity at the early stage of healing and increased expression of Wnt signaling members including Wnt10a, Wnt11, and ß-catenin, which are associated with scar-forming wound repair. In conclusion, pASCs treatment may provide a critical step toward the evaluation of pASCs as therapeutically relevant cells in the context of wound healing.

Perspective on Stem Cell Therapy in Organ Fibrosis: Animal Models and Human Studies.

Tissue fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components that result from the disruption of regulatory processes responsible for ECM synthesis, deposition, and remodeling. Fibrosis develops in response to a trigger or injury and can occur in nearly all organs of the body. Thus, fibrosis leads to severe pathological conditions that disrupt organ architecture and cause loss of function. It has been estimated that severe fibrotic disorders are responsible for up to one-third of deaths worldwide. Although intensive research on the development of new strategies for fibrosis treatment has been carried out, therapeutic approaches remain limited. Since stem cells, especially mesenchymal stem cells (MSCs), show remarkable self-renewal, differentiation, and immunomodulatory capacity, they have been intensively tested in preclinical studies and clinical trials as a potential tool to slow down the progression of fibrosis and improve the quality of life of patients with fibrotic disorders. In this review, we summarize in vitro studies, preclinical studies performed on animal models of human fibrotic diseases, and recent clinical trials on the efficacy of allogeneic and autologous stem cell applications in severe types of fibrosis that develop in lungs, liver, heart, kidney, uterus, and skin. Although the results of the studies seem to be encouraging, there are many aspects of cell-based therapy, including the cell source, dose, administration route and frequency, timing of delivery, and long-term safety, that remain open areas for future investigation. We also discuss the contemporary status, challenges, and future perspectives of stem cell transplantation for therapeutic options in fibrotic diseases as well as we present recent patents for stem cell-based therapies in organ fibrosis.

Effect of Pig-Adipose-Derived Stem Cells' Conditioned Media on Skin Wound-Healing Characteristics In Vitro.

The primary mechanism by which adipose-derived stem cells (ASCs) exert their reparative or regenerative potential relies predominantly on paracrine action. Secretory abilities of ASCs have been found to be amplified by hypoxia pre-conditioning. This study investigates the impact of hypoxia (1% O2) on the secretome composition of pig ASCs (pASCs) and explores the effect of pASCs' conditioned media (CM) on skin cell functions in vitro and the expression of markers attributed to wound healing. Exposure of pASCs to hypoxia increased levels of vascular endothelial growth factor (VEGF) in CM-Hyp compared to CM collected from the cells cultured in normoxia (CM-Nor). CM-Hyp promoted the migratory ability of pig keratinocytes (pKERs) and delayed migration of pig dermal fibroblasts (pDFs). Exposure of pKERs to either CM-Nor or CM-Hyp decreased the levels of pro-fibrotic indicators WNT10A and WNT11. Furthermore, CM-Hyp enhanced the expression of KRT14, the marker of the basal epidermis layer. In contrast, CM-Nor showed a stronger effect on pDFs manifested by increases in TGFB1, COL1A1, COL3A1, and FN1 mRNA expression. The formation of three-dimensional endothelial cell networks was improved in the presence of CM-Hyp. Overall, our results demonstrate that the paracrine activity of pASCs affects skin cells, and this property might be used to modulate wound healing.