Platelet-Derived Microparticels Stimulate Proliferation of Colony-Forming Unit Granulocyte-Macrophage of Umbilical Cord Blood Hematopoietic Stem Cells.

Umbilical Cord blood has become an important source of hematopoietic stem-progenitor cells for transplantation, however hematopoietic recovery after transplantation with umbilical cord blood is slower than with bone marrow or mobilized peripheral blood. Adhesion molecules on hematopoietic cells are involved in hematopoietic cells’ homing, which is considered the most important step of hematological recovery. Some articles indicated that expressions of adhesion molecules on CD34⁺ cells could predict the time to hematopoietic recovery after transplantation with bone marrow and peripheral blood of many adhesion molecules (such as CD62, CXCR4) are significantly lower on umbilical cord blood than on bone marrow. It is a possible reason for the difficulty in hematopoietic recovery after umbilical cord blood transplant. Platelet -derived microparticles (PMPs) are submicroscopic (<1 μm) membrane vesicles released from platelet if they are stimulated with agonists such as thrombin, collagen, or calcium ionophore A23187 or if exposed to high-stress shear forces. PMPs express several platelet-endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib and IaIIa, and P-selectin (CD62P) and several other platelet relevant receptors such as CXCR4 and PAR-1. Some articles indicate that PMPs can affect the function of hematopoietic stem cells by increasing the adhesion of hematopoietic cells to fibrinogen, which suggests that PMP-transferred CD41 antigen plays an important role in this process. PMPs can also increase the survival of human hematopoietic cells including human CD34⁺ clonogenic progenitors. In our research, we observe the function of PMP to affect the cloning efficiency of colony-forming unit granulocyte-macrophage (CFU-GM).

We adopt different concentrations of Thrombin (2U/ml, 1.5U/ml, 1.0U/ml and 0.5U/ml) to activate the platelet and acquire PMPs. Then PMPs were evaluated by using flow cytometry. Based on the result that stimulation of platelets by Thrombin (1U/ml) can acquire the best efficiency of PMPs, we used this concentration in all subsequent experiments. Umbilical cord mononuclear cells (MNCs) were obtained from healthy donors and isolate the MNCs by Ficoll-Hypaque density gradient centrifugation. Briefly, MNCs incubated with or without PMPs cultured in 2.7% methylcellulose. CFU-GM growth was stimulated with 30% umbilical cord serum, rhIL-3 and rh GM-CSF. Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO₂. Colonies were counted under an inverted microscope after 7 or 10 days. The research was divided into four groups: 1. control group; 2. PMPs(10μg/ml); 3. PMPs(50μg/ml); 4. PMPs(100μg/ml). The colony formation was enhanced with PMPs and is dependently stimulated with PMPs. The number of colonies in the group of PMPs(100μg/ml) is more than that of other groups. The number of colonies in control group, PMPs(10μg/ml), PMPs(50μg/ml) and PMPs(100μg/ml) are 57.4±3.2, 65.6±5.6, 77.1±1.7 and 87.8±5.0 per 1×10⁵ respectively. These increases in different groups were statistically significant when compared with control group(p<0.05). To sum up, PMPs can affect the cloning efficiency of CFU-GM of umbilical cord hematopoietic stem cells and the efficiencies are depended on the concentration of PMPs.