Differential adhesion and fibrinolytic activity of mesenchymal stem cells from human bone marrow, placenta, and Wharton's jelly cultured in a fibrin hydrogel

Abstract

Mesenchymal stem cells isolated from different tissues should share associated markers and the capability to differentiate to mesodermal lineages. However, their behavior varies in specific microenvironments. Herein, adhesion and fibrinolytic activity of mesenchymal stem cells from placenta, bone marrow, and Wharton's jelly were evaluated in fibrin hydrogels prepared with nonpurified blood plasma and compared with two-dimensional cultures. Despite the source, mesenchymal stem cells adhered through focal adhesions positive for vinculin and integrin αV in two dimensions, while focal adhesions could not be detected in fibrin hydrogels. Moreover, some cells could not spread and stay rounded. The proportions of elongated and round phenotypes varied, with placenta mesenchymal stem cells having the lowest percentage of elongated cells (~10%). Mesenchymal stem cells degraded fibrin at distinct rates, and placenta mesenchymal stem cells had the strongest fibrinolytic activity, which was achieved principally through the plasminogen-plasmin axis. These findings might have clinical implications in tissue engineering and wound healing therapy.

Keywords: Human mesenchymal stem cells; adhesion; fibrin; fibrinolysis.

Figure 1.

Immunophenotype and differentiation to mesodermal lineages of MSCs. (a) Cells were incubated with labeled antibodies for CD105, CD90, CD73, CD13, HLA-ABC, CD45, CD34, CD31, CD13, and HLA-DR, and analyzed by flow cytometry. Control (dark gray histograms) and positive populations (light gray histograms) are shown. (b) Osteoblastic differentiation was confirmed by alkaline phosphatase staining; glycosaminoglycans in chondroblastic differentiation were stained with Alcian blue, and lipid droplets in adipoblastic differentiation were stained with Oil Red O. Representative experiment. PL-MSCs: placenta mesenchymal stem cells; BM-MSCs: bone marrow mesenchymal stem cells; WJ-MSCs: Wharton’s jelly mesenchymal stem cells.

Figure 2.

Viability of MSCs cultured in fibrin hydrogels and 2D. (a) Cells were stained with LIVE/DEAD kits; viable cells are seen in green, and dead ones in red. MSCs from different tissues showed a viability >90% at 3 days of culture in fibrin and almost 100% as a monolayer on glass coverslips. Images from a representative experiment were taken with an epifluorescence microscope. (b) Graph showing the percentages of viable cells in fibrin hydrogels at 3 days of culture. Bars indicate standard errors of the media of three experiments. There were no statistical differences between MSCs (p < 0.05) (n = 3). PL-MSCs: placenta mesenchymal stem cells; BM-MSCs: bone marrow mesenchymal stem cells; WJ-MSCs: Wharton’s jelly mesenchymal stem cells.

Figure 3.

Temporal course of vinculin and actin distribution in MSCs cultured on 2D. Cells were cultured on glass coverslips for 12 h, 1, 2, and 3 days, fixed and stained for nuclei (blue), F-actin (red), and vinculin (magenta). In each case, vinculin detection in FAs was augmented in a time-dependent manner. Representative confocal micrographs are shown. PL-MSCs: placenta mesenchymal stem cells; BM-MSCs: bone marrow mesenchymal stem cells; WJ-MSCs: Wharton’s jelly mesenchymal stem cells. Bars represent 50 μm.

Figure 4.

Temporal course of vinculin and actin distribution in MSCs cultured in fibrin hydrogels. (a) Cells were cultured in fibrin hydrogels for 0.5 h, 1, 2, and 3 days, fixed and stained for nuclei (blue), F-actin (red), and vinculin (magenta). In each case, two phenotypes can be observed: round-shaped cells positive for vinculin, and elongated cells negative for the marker. Representative confocal micrographs are shown. Bars represent 50 μm. (b) Graph showing the percentages of elongated and round-shaped cells in MSCs cultured in fibrin hydrogels for 2 days. Bars indicate standard errors of the media of three experiments, and 75 cells on average per sample were counted (n = 3). *p = 0.0204, **p = 0.0011, ***p = 0.0007, and ****p < 0.0001. PL-MSCs: placenta mesenchymal stem cells; BM-MSCs: bone marrow mesenchymal stem cells; WJ-MSCs: Wharton’s jelly mesenchymal stem cells.

Figure 5.

Presence of integrin αV in MSCs cultured in 3D fibrin hydrogel and 2D. (a) Integrin αV detection in MSCs cultured as monolayer on glass coverslips for 48 h. Cells were fixed and stained for nuclei (blue), actin (red), vinculin (magenta), and integrin αV (green). In each case, integrin αV was detected in FAs, where it partially colocalized with F-actin and vinculin. (b) Integrin αV detection in MSCs cultured in fibrin hydrogels for 48 h. Cells were fixed and stained for nuclei (blue), actin (red), vinculin (magenta), and integrin αV (green). Integrin αV was detected in some elongated and round-shaped cells; 3D reconstructions are displayed (see Supplemental Videos S1–S3). (c) CD105 marker in WJ-MSCs cultured in fibrin. Cells were fixed and stained for nuclei (blue), actin (red), vinculin (magenta), and CD105 (green). CD105 was detected in elongated and round-shaped cells. This was also observed in PL-MSCs and BM-MSCs (data not shown). (a)–(c), representative confocal micrographs are shown. PL-MSCs: placenta mesenchymal stem cells; BM-MSCs: bone marrow mesenchymal stem cells; WJ-MSCs, Wharton’s jelly mesenchymal stem cells. Bars represent 50 μm.

Figure 6.

Fibrinolysis by MSCs. (a) Cells seeded in fibrin gels generated hydrolysis at 48 h of culture. Images show degraded zones (*) and cells (arrows). Bars represent 50 μm. (b) Cells seeded in fibrin gels in the presence or absence of inhibitors—Ap (200 μg/mL), TA (400 μg/mL), BB-94 (5 μM), and their combinations—generated distinct levels of hydrolysis at 7 days of culture. Bars represent 3 mm. (c) Semi-quantification of total area of degraded zones done by MSCs in the presence or absence of inhibitors. Bars indicate standard errors of two experiments (n = 2). *p = 0.0104 and ****p < 0.0001. PL-MSCs: placenta mesenchymal stem cells; BM-MSCs: bone marrow mesenchymal stem cells; WJ-MSCs: Wharton’s jelly mesenchymal stem cells; TA: tranexamic acid; Ap: aprotinin.

Figure 7.

MSCs adhesion to fibrin hydrogels and fibrinolysis effect. MSCs from the three tissues cultured in fibrin hydrogels exhibited elongated and round phenotypes, with actin in filaments or aggregates, respectively. Vinculin and integrin αV were not necessary for adhesion, as they were absent in FAs, and only some cells were positive for both proteins. Proportions of elongated and round-shaped cells varied between the three types of MSCs accordingly to their capabilities to degrade fibrin hydrogel. PL-MSCs were rounded in >90%, degraded the hydrogel completely, and generated a monolayer with FAs rich in vinculin (see Supplemental Figure S1), and were positive probably for integrin αV (as it was shown in 2D cultures, see Figure 5). BM-MSCs and WJ-MSCs degraded fibrin in a limited way, and after 3 days they still gathered inside it. In all cases, aprotinin and tranexamic inhibited fibrinolysis more than BB-94, meaning that it was done principally via the plasminogen–plasmin axis. uPA: urokinase plasminogen activator; tPA: tissue plasminogen activator; PAI-1: plasminogen activator inhibitor 1; MMPs: metalloproteases; TIMPs: tissue inhibitors of metalloproteases.