Influence of Medium Components on Ex Vivo Megakaryocyte Expansion

Abstract

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study, we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion, we used an in vitro liquid culture system. For this purpose, CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes, implying the presence of inhibitory factors in plasma. However, some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma, higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore, Cellgro medium is not the best alternative medium. In cultures with Stemspan medium, higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides, AF13948 and PK1M, were tested for their ability to replace Tpo. In cultures with AF13948, comparable numbers of megakaryocytes were obtained as in the presence of Tpo, but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation, but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol, it is worthwhile to test several media, because the number of megakaryocytes differs widely with the medium used.

Bone marrow niche-inspired, multiphase expansion of megakaryocytic progenitors with high polyploidization potential

“…3 Mk progenitors per input HPSC in cultures with 100 ng/mL Tpo and 10 ng/mL IL-1β [41]. However, many groups have reported fewer than 2.5 CD34 + CD41 + cells per input CD34 + cell [13, 17, 42–45]. For example, De Bruyn et al reported 1.8 Mk progenitors per input HPSC in cultures with 100 ng/mL Tpo, 100 ng/ml FL, 10 ng/ml IL-6, and 10 ng/ml IL-11 [13].…”

“…This level of Mk production from HSPCs derived from mPB donors is rare, although one donor sample from the present study produced 30 Mks per input HSPC. Most groups report fewer than 7 Mks per mPB CD34 + cell [13, 17, 32, 41, 42, 45–48]. Further, relatively few groups have reported whether the expanded Mks attained high-ploidy.…”

Comparison of the biological activities of anagrelide and its major metabolites in haematopoietic cell cultures

“…To examine the effect of test compounds on the expansion of CD34 + cells resulting from TPO stimulation, these cells were grown under conditions which support the proliferation of MK progenitors in serum‐free medium ( Lam et al ., 2001 ; van den Oudenrijn et al ., 2001 ). Freshly isolated cells were cultured with 40 ng ml −1 recombinant human TPO (R&D Systems, Abingdon, U.K.) in Stemspan™ medium (Stem Cell Technologies, London, U.K.) which consisted of Iscove's‐modified Dulbecco's medium (IMDM) supplemented with 1% bovine serum albumin, 10 μ g ml −1 recombinant human insulin, 200 μ g ml −1 iron‐saturated transferrin, 0.1 m M 2‐mercaptoethanol and 2 m M glutamine.…”

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

“…CD34 + HSPCs from G-CSF-mobilized peripheral blood (mPB) or umbilical cord blood (CB) have been used to produce Mks by many investigators. Culture conditions that result in ‡ 50% Mks typically yield 5-10 Mks per input mPB CD34 + cell [18][19][20][21][22] ; producing one transfusion dose under these conditions would require *250 million CD34 + cells. CB CD34 + cells typically yield 10-fold more Mks per CD34 + cell than mPB CD34 + cells.…”