Coagulopathy As a Dose-Limiting Toxicity in Systemic Administration of Mesenchymal Stem Cells (MSCs): Relative Safety of Bone Marrow Versus Adipose Derived MSCs

Introduction: Systemic administration of mesenchymal stem cells (MSCs) is an option that allows MSCs to interact with circulatory immune cells, or to deliver MSCs into multiple traumatic sites that are not reachable through regional delivery. The number of MSCs per injection also needs to be determined in order to achieve the maximal beneficial effect, and optimize the ratio of MSCs to target cells. However, MSCs express tissue factor, which is known to activate coagulation, which could potentially cause inappropriate clot formation, counteracting the beneficial effect of MSCs on tissue regeneration and immunomodulation at the site of injury. In this study, we sought to determine the maximum dose of MSCs that can safely be used for systemic administration by measuring their impact on coagulation in rats.

Method: Bone marrow and adipose derived MSCs (BMSC and AMSC) were isolated from bone (femur and tibia) and visceral fat tissue, respectively, in normal young Sprague Dawley (SD) rats. Both BMSCs and AMSCs were cultured and passaged using Dulbecco's Modified Eagle's Medium (DMEM) with 20% fetal bovine serum. MSCs at passage 2-5 were labeled with green fluorescent chloromethyl derivatives of fluorescein diacetate (CMFDA), resuspended in normal saline with anticoagulant (Citrate Phosphate Dextrose (CPD)), and infused into the SD rats through the femoral vein at doses of 2.5, 5, 10, 20, and 40 million/kg under anesthesia. Citrated whole blood was collected at the base line, immediately after infusion, 1 hour, and 3 hours after infusion of MSCs. The prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen were measured by hemostasis analyzer (ST4); coagulation properties and platelet aggregation (response to ADP (Adenosine Diphosphate) and collagen) were measured by hemostasis analyzer, rotational thromboelastometry (ROTEM) and impedance aggregometry (Multiplate), respectively. The MSCs in circulation were measured by flow cytometry. The MSCs in tissue were determined by histology.

Results: At the dose of 20 million/kg, rats survived with BMSCs, but died with AMSCs (n=3 per group). Two of three rats survived with infusion of BMSCs at 40 million/kg. The platelet counts were significantly reduced at 1hr after MSC infusion (%change to baseline: AMSCs: -4±0; -24±3; -39±8 % at 2.5, 5 and 10 million/kg, respectively; BMSCs: -3±0, -4±3, -18±4, -19±6, -63±5 % at 2.5, 5, 10, 20 and 40 million/kg, respectively). Both PT and aPTT were significantly prolonged (PT: 35±4(AMSC) vs. 9±3(BMSC)% increased; aPTT 81±6(AMSC) vs. 17±3(BMSC)% increased), and fibrinogen levels significantly declined (-39±8(AMSC) vs. -18±4(BMSC)% decreased) after 10 million/kg AMSC or BMSC infusion. In comparison to the baseline, clotting time (CT) was significantly prolonged (AMSCs: 124±14 (vs. 68±6); BMSCs: 82±8 (vs. 57±4) seconds), and maximum clot firmness (MCF) was significantly reduced (AMSC: 39±3 (vs. 58±2) mm); BMSCs: 38±7 (vs. 62±4) mm) at 1hr after infusion of AMSCs at 10 million/kg or BMSCs at 20 million/kg respectively. Platelet aggregation (area under the curve (mm²)) significantly declined compared to the baseline after infusion of 10 million/kg AMSCs (ADP: 10±4 (vs. 75±7.0); Collagen: 4±4 (vs. 74±5)) or 20 million/kg BMSCs (ADP: 20±15 (vs. 89±12); Collagen: 36±18 (vs.102±10)). There were few circulating AMSCs or BMSCs found after 1 and 3 hours after infusion. The MSCs were found mostly in the lung, as determined by histology.

Conclusion: AMSCs and BMSCs are not maintained in the circulation after systemic administration. However, infusion of MSCs has a significant impact on the hemostatic system, as shown by prolongation of PT, aPTT, and CT; and a decrease in fibrinogen levels, platelet counts, platelet function, and clot firmness. These data suggest that systemic administration of MSCs has a potential risk of causing consumptive coagulopathy, which may be initiated by the tissue factor expression of MSCs. This study also suggests that BMSCs have less impact than AMSCs on hemostasis, and BMSCs at the dose of 5 million/kg or less has the least risk of causing coagulopathy. Future studies are necessary to investigate the appropriate methods of down-regulating the procoagulant properties of MSCs prior to systemic administration in order to minimize the potential risk of coagulopathy.