Fiche publication
Date publication
mai 2021
Journal
Journal of visualized experiments : JoVE
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GACHET Christian
Tous les auteurs :
Pongerard A, Mallo L, Gachet C, de La Salle H, Lanza F, Strassel C
Lien Pubmed
Résumé
The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.
Référence
J Vis Exp. 2021 May 20;(171):
