Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.

Adipose-derived stem cells (ASCs) isolated from domestic animals fulfill the qualitative criteria of mesenchymal stem cells, including the capacity to differentiate along multiple lineage pathways and to self-renew, as well as immunomodulatory capacities. Recent findings on human diseases derived from studying large animal models, have provided evidence that administration of autologous or allogenic ASCs can improve the process of healing. In a narrow group of large animals used in bioresearch studies, pigs and horses have been shown to be the best suited models for study of the wound healing process, cardiovascular and musculoskeletal disorders. To this end, current literature demonstrates that ASC-based therapies bring considerable benefits to animal health in both spontaneously occurring and experimentally induced clinical cases. The purpose of this review is to provide an overview of the diversity, isolation, and characterization of ASCs from livestock. Particular attention has been paid to the functional characteristics of the cells that facilitate their therapeutic application in large animal models of human disease. In this regard, we describe outcomes of ASCs utilization in translational research with pig and horse models of disease. Furthermore, we evaluate the current status of ASC-based therapy in veterinary practice, particularly in the rapidly developing field of equine regenerative medicine. In conclusion, this review presents arguments that support the relevance of animal ASCs in the field of regenerative medicine and it provides insights into the future perspectives of ASC utilization in animal husbandry.Graphical abstract

Recent studies have shown that the transcription factor Foxn1, which is expressed in keratinocytes, is involved in the skin wound healing process, yet how Foxn1 functions remains largely unknown. Our latest data indicate that Foxn1 drives skin healing via engagement in re-epithelization and the epithelial-mesenchymal transition (EMT) process. In the present study, 2D-DIGE proteomic profiling analysis of in vitro cultured keratinocytes transfected with adenoviral vector carrying Foxn1-GFP or GFP alone (control) revealed forty proteins with differential abundance between the compared groups. Among the proteins with Foxn1-dependent expression, several enable adaptation to hypoxia. Subsequent experiments revealed that hypoxic conditions (1% O₂) stimulate endogenous and exogenous (transfected Ad-Foxn1) Foxn1 expression in cultured keratinocytes. A proteomics analysis also identified proteins that can act as a factors controlling the balance between cell proliferation, differentiation and apoptosis in response to Foxn1. We also showed that in C57BL/6 keratinocytes, the stimulation of Foxn1 by hypoxia is accompanied by increases in Mmp-9 expression. These data corroborate the detected co-localization of Foxn1 and Mmp-9 expression in vivo in post-wounding skin samples of Foxn1::Egfp transgenic mice. Together, our data indicate that Foxn1 orchestrates cellular changes in keratinocytes in both physiological (self-renewal) and pathological (skin wound healing) contexts.

Since there are limited studies analyzing the impact of age, sex and obesity on cutaneous repair, the current study evaluated excisional skin wound healing as a function of age, sex and diet in C57BL/6 mice subjected to either low (LFD) or high (HFD) fat diet. Older mice accumulated increased body fat relative to younger mice under HFD. Skin wound healing at particular stages was affected by age in the aspect of Tgfβ-1, MCP-1, Mmp-9 and Mmp-13 expression. The most profound, cumulative effect was observed for the combination of two parameters: age and sex. While skin of younger males displayed extremely high collagen 1 and collagen 3 expression, younger females showed exceptionally high Mmp-13 expression at day 3 and 7 after injury. Diet as a single variable modified the thickness of dermis due to increased dermal White Adipose Tissue (dWAT) accumulation in mice fed HFD. The combination of age and diet affected the re-epithelialization and inflammatory response of injured skin. Overall, our data indicate that age has the most fundamental impact although all components (age, sex and diet) contribute to skin repair.

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin’s pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor β family (TGFβ) expression showed significantly higher levels of Tgfβ3 transcript between nude and Balb/c mice but no differences were detected for Tgfβ1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFβ3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFβ3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.