Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins

Abstract

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Figure 1

Isolation and characterization of cMSCs. (a) cMSCs were isolated from subcutaneous adipose tissue and plated (magnification 10x). (b) cMSCs were grown in DMEM-F12 20% FBS and counted at 24 h, 48 h, and 72 h. The data obtained represent the mean ± SD of three independent experiments. The statistical analysis was calculated by the one-way ANOVA test. Asterisks denote statistical significance versus time point zero (^∗p < 0.05, ^∗∗∗∗p ≤ 0.0001). (c) Representative quadrant dot plots from FACS analysis of cMSCs stained for CD90, CD44, CD105, and CD45 antigens. (d) Representative microscopic images (magnification 20x) from Alizarin Red staining (ARS) for calcium accumulation detection in undifferentiated and osteoblast-like differentiated cells. (e) Representative microscopic images (magnification 20x) of undifferentiated and adipocyte-differentiated cells.

Figure 2

hPRP effect on MHC-I genes. mRNA levels of DLA12 and DLA64 were determined by qPCR analysis of total RNA isolated from cMSCs treated with 10% hPRP for 48 h. The average expression value of DLA12 (a) and DLA64 (b) in cMSCs treated with DMEM-F12 0.25% BSA (control cells) was used as a reference sample and GAPDH as a housekeeping gene.

Figure 3

hPRP effect on cMSC proliferation and migration. (a) cMSCs were treated with 10% hPRP-CM (obtained in 0.25% BSA medium, as described in Material and Methods) for 48, 72, 96, and 120 hours. As a control, cells were seeded in DMEM F12 (1 : 1) with 0.25% BSA or with 20% FBS (standard growth conditions). Cell viability was analyzed by the MTT assay. (b) cMSCs were serum-starved for 18 h and then seeded in the upper chamber of a transwell culture system. hPRP gel (10% vol/vol in DMEM F12 with 0.25% BSA) was added in the lower chamber in the presence of 0.25% BSA medium for 48 h. As a control, DMEM F12 (1 : 1) without serum supplementation (0.25% BSA) or with 20% FBS (standard growth conditions) was added to the lower compartment. Cells that migrated across the filter were determined by crystal violet staining, as described in Materials and Methods. Bars show the fold-over control represented by cells in 0.25% BSA. Before crystal violet elution, cells were photographed (magnification 10x). (a, b) The data represent the mean ± SD of three independent experiments. ^∗ denotes statistical significance versus cells in 0.25% BSA medium (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001); # denotes statistical significance versus cells in 20% FBS medium (###p < 0.001).

Figure 4

hPRP effect on cMSC AQPs. cMSCs were treated with 10% hPRP-conditioned medium for 24 h. Cell lysates (20 μg protein/sample) were blotted with AQP1 (a) and AQP5 (b) antibodies. To ensure equal protein transfer, membranes were blotted with vinculin antibodies. Filters were revealed by enhanced chemiluminescence (ECL) and autoradiography. The autoradiographs shown are representative of three independent experiments. Densitometric analyses have been performed on autoradiographs. Bars show the ratio between AQPs and vinculin protein levels as a fold-over control (cells in 0.25% BSA). The data were analyzed using the Mann–Whitney test (^∗p < 0.05).

Figure 5

Effect of TEAC inhibitor. (a) cMSCs were treated with three different concentrations of TEAC (10 μM, 50 μM, and 100 μM) in DMEM F12 0.25% BSA medium. Cell viability was analyzed by the sulforodamine assay. Bars show the viability compared to control cells (those cultured in 0.25% BSA). The data obtained represent the mean ± SD of four independent experiments. (b) cMSCs were seeded in the upper chamber of a transwell culture system in the presence of 10% hPRP in the lower chamber with or without 100 μM TEAC. Cells that migrated across the filter were determined by crystal violet staining. Bars show the fold-over control (cells in the presence of 10% hPRP). Before crystal violet elution, cells were photographed (magnification 10x). The data represent the mean ± SD of three independent experiments (^∗p < 0.05).