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• W2519133068 abstract "Abstract Platelet transfusions are widely used for patients with severe thrombocytopenia. There are, however, practical problems in the current donor-dependent platelet transfusions, such as the limited supply and risk of serious immune reactions. Thus, the development of new strategies for generating platelets for transfusion is crucial. Platelets have been differentiated from hematopoietic stem cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, NF-E2-transduced fibroblasts, and preadipocytes. Here, among these cells preadipocytes, especially in the subcutaneous adipose tissue, could be ideal candidate cells for manufacturing megakaryocytes (MKs) and platelets, because (1) they are relatively easy to obtain large quantities and have ability to proliferate in vitro, (2) their differentiation does not require gene transfer, as they possess genes in relation to megakaryopoiesis and thrombopoiesis, such as p45NF-E2 and c-mpl, and (3) they differentiate into MKs and platelets using an endogenous thrombopoietin. Thus, to clarify the usefulness of preadipocytes as a donor-independent source for platelet transfusion, we compared both number and function between platelets derived from mouse subcutaneous preadipocytes and those from bone marrow mononuclear cells (BMMNCs), the established cell source for manufacturing platelets. First, BMMNCs were not feasible for their expansion in vitro and therefore the cells were directly seeded in MK lineage induction media. In contrast, preadipocytes were to be passaged 6 times without any morphological changes, and then cultured in MK lineage induction media for their differentiation into platelets. Thus, as assessed by CD41-positive platelet-sized cells, 106.2±5.0 ×105 or 3.9±1.0 ×105 platelets were obtained from 106 preadipocytes or 106 BMMNCs, respectively (p<0.01). To next analyze platelet function, the binding of Alexa Fluor 488-labaled fibrinogen to CD41-positive platelet-sized cells, as assessed by mean fluorescence, was analyzed in the presence of ADP/PAR4-activating peptide, and the binding was higher in preadipocyte-derived platelets (14.0±1.5) than in BMMNC-derived platelets (7.0±1.3, p=0.017). Furthermore, to examine the incorporation of these platelets into a thrombus under flow conditions, we performed the quantitative evaluation of the cells in the thrombus formation process using the Total Thrombus-formation Analysis System (T-TAS®) (Blood 2012; 120: 3812). We used platelets released from each preadipocyte- and BMMNC-derived MKs in vivo. Briefly, MK-sized cells were obtained by a 2-step BSA gradient, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then infused into irradiated thrombocytopenic mice (7 days after exposure to 2.0 Gy). After 90 min of infusion, whole blood samples were obtained from these mice and were shown to contain similar number of CFSE+/CD41+ platelet-sized cells; 1.8±0.4 ×104 for preadipocyte-derived cells and 1.9±0.2 ×104 for BMMNC-derived cells (p=0.9135). Subsequently, the samples were perfused on a collagen-coated chip, followed by assessment using T-TAS®, and the result showed that the frequency of incorporation of the cells into the thrombus formation was higher for preadipocyte-derived cells (77.8±5.1%) than for BMMNC-derived cells (52.5±5.3%, p=0.0257). And we also found that the frequencies were similar when 3-fold amounts of BMMNC-derived cells were used in this perfusion study. Here our microarray analyses showed that plasminogen activator inhibitor-1 (PAI-1), a well-known enhancer for platelet function, was secreted into the supernatant during MK differentiation process. Therefore, to elucidate the possibility that PAI-1 is associated with the findings as described above, the levels of PAI-1 in the supernatant were measured on Day 4 during MK differentiation, and were shown to be 3.3±0.1 ng/mL or 0.09±0.01 ng/mL for preadipocyte- or BMMNC-derived cells, respectively. Together, our findings provided the first evidence showing that preadipocytes produce a greater number of platelets than BMMNCs and their derived platelets also have more useful physiological function than BMMNC-derived platelets, allowing for the usage of fewer platelets in platelet transfusion. Disclosures: No relevant conflicts of interest to declare." @default.
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• W2519133068 date "2013-11-15" @default.
• W2519133068 modified "2023-09-28" @default.
• W2519133068 title "Subcutaneous Preadipocytes Are Ideal Candidate To Produce A Large Number Of Functional Platelets In a Culture System" @default.
• W2519133068 doi "https://doi.org/10.1182/blood.v122.21.3271.3271" @default.
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