Secreted factors from equine mesenchymal stromal cells diminish the effects of TGF‐β1 on equine dermal fibroblasts and alter the phenotype of dermal fibroblasts isolated from cutaneous fibroproliferative wounds

Abstract

The prevalence of cutaneous fibroproliferative disorders (CFPDs) is high and almost exclusively occurs in humans (keloids and hypertrophic scars) and horses (exuberant granulation tissue), making the horse a valuable translational model for studies on prevention and treatment of human CFPDs. CFPDs arise as a result of dysregulated wound healing characterized by persistently high levels of cytokines, such as transforming growth factor beta 1 (TGF-β1), that contribute to excessive extracellular matrix deposition, and the physical disorganization of dermal fibroblasts (DF). The mesenchymal stromal cell (MSC) secretome, consisting of all factors secreted by MSC, has been shown to promote normal wound healing in both humans and horses, but its potential to treat CFPDs remains largely unexplored. Therefore, the objective of this study was to examine the effects of the equine MSC secretome on equine DF influenced by cytokines that contribute to the development of CFPDs. First, primary equine DF were treated with TGF-β1 in vitro in the presence or absence of MSC secreted products. We found that MSC secreted products could block TGF-β1-induced changes in DF morphology, proliferation rate, gene expression, and contractile-capacity. We then isolated primary DF from equine exuberant granulation tissue, to evaluate the potential of the MSC secretome to alter the phenotype of cells derived from a complex CFPD environment. These results showed that MSC secreted factors did not change proliferation or migration of these cells, but did lead to changes in expression of genes and proteins involved in extracellular matrix remodeling and did affect contractile capacity. These results warrant future studies designed to evaluate the potential of the MSC secretome to minimize the pathologies associated with CFPD in vivo.

Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds

“…Taken together, our group focuses on the potential of equine MSC to be used as a therapy for skin wounds and the data generated in this study suggests that in addition to positively affecting resident skin cells, as we have demonstrated previously [25, 26, 31], equine MSC may also improve cutaneous wound healing by reducing the bacterial load in wounds. Next, we intend to evaluate the efficacy of equine MSC in vivo by assessing how these cells promote skin wound healing and affect bacterial burden, in both acute and chronic wounds.…”

“…Western blot analyses were performed, exactly as previously described [26], using the same Ab that were used to block AMP activity in MSC CM, diluted 1:500, followed by horseradish peroxidase (HRP)-conjugated goat anti rabbit secondary Ab (Jackson ImmunoResearch Labs, West Grove, PA, USA), diluted 1:20,000.…”

“…Recent data show that paracrine signaling is the primary mechanism by which MSC contribute to tissue repair [23, 24]. Our laboratory has previously demonstrated that equine MSC conditioned medium (CM), comprising all factors secreted by MSC, can increase equine dermal fibroblast migration and block the effects of transforming growth factor beta-1 on equine dermal fibroblasts, providing the rationale for their potential in cutaneous wound management [25, 26]. The potential of equine MSC to contribute to wound healing by reducing bacterial loads has not been explored to date, although this seems achievable based on previous reports showing that human MSC possess antimicrobial properties such as killing Escherichia coli ( E. coli ) in rodent lung infection models, as well as reducing overall bacterial loads in septic mice [27–29].…”

Single-cell RNA sequencing of equine mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility

“…SYBR green-based RT-PCR was performed to determine fold change in transcripts of interest, and data were analyzed, as previously described [41,42]. The previously validated reference gene beta-2-microglobulin (BM2) was used to normalize samples [43] and all samples, were run in triplicate. Genes and primer sequences are listed in Table 1.…”

“…To asses proliferation, a 5-Bromo-2′-deoxyuridine (BrdU) incorporation ELISA (Abcam) was used, as previously described [43]. Cell adhesion was determined using a microcentrifugation assay, as previously described [42].…”