Adipose mesenchymal stromal cells isolated from type 2 diabetic patients display reduced fibrinolytic activity

Abstract

Stem cells have been successfully used for the treatment of critical limb ischemia (CLI). We conducted a clinical trial to determine the feasibility of using autologous adipose-derived mesenchymal stromal cells (AdMSCs) for the treatment of CLI. Unexpectedly, two diabetic patients developed peripheral microthrombosis. This adverse effect, which contrasts with the reported antithrombotic properties of MSCs, may stem from the diabetic environment that alters the fibrinolytic activity of AdMSCs, thereby increasing the probability of developing thrombosis. Here, we confirm this premise by demonstrating that diabetic AdMSCs cultured in the presence of blood sera expressed and released higher levels of plasminogen activator inhibitor type 1, reduced levels of tissue plasminogen activator, and lower d-dimer formation compared with nondiabetic AdMSCs. Thus, to establish an appropriate cell therapy for diabetic patients, we recommend including new preclinical safety tests, such as the d-dimer and/or the tissue plasminogen activator-to-plasminogen activator inhibitor type 1 ratio tests, to assess fibrinolytic activity of cells before implantation.

Figures

FIG. 1.

AdMSCs derived from diabetic patients display reduced and serum-independent fibrinolytic activity. Expression analysis of tPA and PAI-1 I shown in AdMSCs isolated from AdMSCs, AdMSC-D, and AdMSC-ND and cultured in the presence of GS (control), hBS, hBS-D, and hBS-ND. Detection of tPA mRNA expression (A). tPA protein release shows that the blood sera, independent of its origin, inhibited tPA expression in AdMSC-D (B). PAI-1 mRNA expression (C). D: PAI-1 protein release. Note that AdMSC-D secreted the highest quantity of PAI-1. Relative mRNA expression indicates the ratio between specific gene and housekeeping genes. Values are normalized to the expression in no stimulated AdMSCs, which was arbitrarily set as 1. E: Hematoxylin and eosin staining shows AdMSCs-mediated lysis of the fibrin gel. The cell is surrounded by a clearing zone, except for AdMSC-D, which displays hampered capacity to degrade fibrin gel independently of human blood serum used. Scale bars: 50 µm. F: Free cell/area quantification from hematoxylin-eosin staining of arbitrary images. G: In quantification of d-dimer production of AdMSCs cultured in fibrin clot, AdMSC-D displayed blunted capacity to generate d-dimers independently of serum used. Data are presented as mean values ± SEM (n = 4). Statistical significance: *P ≤ 0.05, **P ≤ 0.01 for AdMSC-D compared with AdMSC-ND.