Abstract
Platelet transfusion is essential for the treatment of thrombocytopenia. Platelet supply depends on blood donation and the ability to store platelets before use is limited by a short shelf-life. Furthermore, such supplied-platelets have the risk of bacterial or viral infection. Ex vivo platelet production could overcome these problems. Therefore, many methods of ex vivo platelet production from some stem cell sources such as umbilical cord blood (UCB) stem cells, human embryonic stem cells and induced pluripotent stem cells have been developed. However, the platelet yields achieved by these methods have not been sufficient for clinical use. The processes of platelet production are thought to involve differentiation into megakaryocytes (Mks) from hematopoietic stem cells, Mk polyploidization, proplatelet formation, and finally platelet release. Large-scale ex vivo production of functional platelets requires a system to control all these steps. Hematopoietic progenitor cell and Mk levels can be increased by the numerous hematopoietic growth factors; however, factors to stimulate proplatelet formation in Mks have not been identified.
To find factors to stimulate proplatelet formation in Mks, we evaluated the effects of various conditioned media using Mks induced from UCB-CD34+ cells. The supernatants of COS cells transfected with cDNA coding for non-muscle cofilin stimulated proplatelet formation in Mks, whereas those of mock-transfected COS cells did not. Cofilin is an actin-binding protein; recently, endogenous non-muscle cofilin was reported to be associated with proplatelet formation in Mks, but that activity has not been reported for exogenous cofilin. The effects of cofilin on proplatelet formation in Mks in the presence or absence of TPO were compared with that in the presence of TPO alone. Cofilin alone increased the number of proplatelet-bearing Mks and enhanced proplatelet extension compared to TPO alone; moreover, a combination of cofilin and TPO had synergistic effects on both activities.The influences of cofilin on megakaryopoiesis from UCB-CD34+ cells were compared with those of TPO. The addition of cofilin to serum-free culture containing SCF and Flt3-L increased the size of both Mks and Mk colonies compared to SCF/Flt3-L/TPO. Next, the effects of cofilin on the platelet production in the serum-free culture system containing SCF and Flt3L were evaluated; the addition of coflin dramatically increased the number of platelets compared to that for SCF/Flt3-L/TPO. Thus, we established a suitable system for ex vivo platelet production by using cofilin. To determine whether the platelets produced using this system were functional; platelet activation in response to in vitro activation with thrombin was tested. Thrombin induces platelet granule secretion, resulting in CD62P (P-selectin) expression on the platelets surface membrane. The expression of CD62P was increased after thrombin stimulation of the platelets produced in cofilin culture. These results indicate that the platelets produced with cofilin were functional.
We show here that exogenously adding cofilin to the serum-free culture system enhanced proplatelet formation in Mks, resulting in increased platelet yields. At present, many methods are under study for ex vivo platelet production for clinical use; we believe that our studies will provide the important information for the improvement of these methods.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.