Estudio in vitro del efecto de las células madre mesenquimales de líquido amniótico sobre la función plaquetaria

Résumé

Introduction: Mesenchymal stem cells (MSCs) obtained from extraembryonic tissues, represent a new source of stem cells for use in cell therapy. They are exempt from the ethical problems and tumorigenic potential associated with embryonic stem cells. Moreover, with respect to adult stem cells, they have a greater capacity for proliferation and differentiation. One of the sources of MSCs is amniotic fluid, which have shown to have excellent immunomodulatory, anti-inflammatory properties, as well as low immunogenicity and teratogenicity in in vivo transplants. Recently, side effects have been described, mainly thrombotic phenomena were administered intravenously. These adverse effects, have been related to a greater expression of tissue factor, which would activate the extrinsic coagulation pathway, ultimately producing thrombin. It has been seen how MSCs interact with other cells, including platelets, elements with a fundamental role in hemostasis. Objectives: The main objective is to evaluate the in vitro effect of amniotic fluid mesenchymal stem cells (MSCs-LA) on platelet function. As secondary objectives we consider the isolation, culture, characterization of MSCs-LA obtained from term births, analyze the effect of the MSCs -LA on the aggregation and adhesion, evaluate the effect of the MSCs-LA on the expression of activation markers platelet and the study of the effect of the MSCs -LA on the cytoplasmic levels of platelet calcium. Material and methods: MSCs were obtained from amniotic fluids (LA) of scheduled cesarean deliveries at term. Later they were cultivated in Amniomed medium, monitoring the culture every 48-72 hours. After 30 days, their capacity for differentiation into chondrocytes, adipocytes and osteocytes was evaluated, as well as their identification by phenotypic characterization. Likewise, the molecular analysis of the MSCs-LA was carried out. Regarding the analysis of the effect of the MSCs -LA on the platelet function, the classic PFA-100 and Multiplate trials were carried out. The effect of MSCs-LA on platelet aggregation, on the expression of platelet activation markers and on the cytoplasmic levels of platelet calcium was also analyzed by flow cytometry. Results: The cultures of MSCs-LA showed high proliferation, differentiation capacity and expressed the mesenchymal markers CD105+, CD90+ and CD73+ and the factors of coagulation and adhesion: factor VIII, von Willebrand factor, tissue factor and podoplanin, being negative for the hematopoietic markers CD45- and CD34-. No differences were observed by PFA-100 analyzer. By the other way, the Multiplate system and the flow cytometric analysis did show changes in aggregation and platelet activation (expression of P-selectin) both basally and in the presence of agonists. When evaluating platelet calcium by flow cytometry, we observed how the presence of MSCs-LA produces a slight increase in cytoplasmic calcium in isolated platelets, maintained only in the presence of extracellular calcium. On the contrary, platelets that interact with MSCs-LA showed a progressive and significant increase in cytoplasmic calcium. heparin potentiate the inhibition of the aggregation and activation in platelets. Conclusions: The amniotic fluid obtained from term deliveries is an adequate source for obtaining MSCs. MSCs-LA induce changes in platelet function, regardless of their procoagulant effects.