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  • Ex vivo differentiation of cord blood stem cells into megakaryocytes and platelets.

Ex vivo differentiation of cord blood stem cells into megakaryocytes and platelets.

Methods in molecular biology (Clifton, N.J.) (2012-11-28)
Nicolas Pineault, Amélie Robert, Valérie Cortin, Lucie Boyer
ABSTRACT

Megakaryocytes (MK) are hematopoietic cells present in the bone marrow that are responsible for the production and release of platelets in the circulation. Given their very low frequency (<1%), human MK often need to be derived in culture to study their development or to generate sufficient material for biological studies. This chapter describes a simplified 14-day culture protocol that efficiently leads to the production of MK and platelets from cord blood enriched progenitor cells. A serum-free medium is suggested for the growth of the CB cells together with an optimized cytokine cocktail developed specifically for MK differentiation, expansion, and maturation. Methodologies for flow cytometry analysis, MK and platelets estimation, and MK progenitor assay are also presented.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, ACS reagent, 99.0-101.0%
Sigma-Aldrich
Ribonuclease A from bovine pancreas, for molecular biology, ≥70 Kunitz units/mg protein, lyophilized
Sigma-Aldrich
Theophylline, anhydrous, ≥99%, powder
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, Type II-S, lyophilized powder, Protein ≥80 %, ≥2,000 units/mg protein