MK differentiation in culture. CD34 + -enriched cells from leukofilters (TACSI, Terumo BCT, Zaventem, Belgium) were expanded using a previously described two-phase optimized protocol 11 . Briefly, the filter extract was enriched in CD34 + cells by magnetic activated cell sorting (CD34 MicroBead Kit UltraPure, Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then seeded in StemSpan serum-free expansion medium (SFEM) supplemented with 20 µg/mL human low-density lipoprotein and a cocktail of cytokines (CC220, Stemcell Technologies, Vancouver, BC, Canada) and with 1 µM SR1 (Stemcell Technologies). On day 7, the cells were open Scientific RepoRtS | (2020) 10:914 | https://doi.org/10.1038/s41598-020-57754-9www.nature.com/scientificreports www.nature.com/scientificreports/ harvested, washed, seeded in StemSpan SFEM containing 1 µM SR1, 50 ng/mL TPO and 20 µg/mL human low-density lipoprotein and cultured for an additional 6 days. The cultures were incubated at 37 °C under normoxic conditions and a 5% CO 2 atmosphere. platelet isolation. Cultured Platelets were harvested after addition of 0.5 µM PGI 2 and 0.02 U/mL apyrase to the culture plates followed by successive pipetting. The platelet-like particles were then centrifuged and resuspended in Tyrode's albumin buffer as previously described 11 . platelet ultrastructure and morphology. Transmission electron microscopy. Cultured platelets or native platelets were fixed in 2.5% glutaraldehyde and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and examined under a JEol 2100-plus (Jeol, Japan) 12 .Confocal microscopy. After fixation in paraformaldehyde, platelets were cytospun, permeabilized with 0.1% Triton X-100 in PBS and incubated sequentially for 30 min with an anti-ß1-tubulin mAb (1:400, 1 μg/mL, Eurogentec, Liège, Belgium) followed by a secondary GAM-488 antibody (10 µg/mL) and an anti-GPIIbIIIa mAb (Alma.17-647, 10 µg/mL) in PBS containing 1% BSA. The cells were then embedded in Mowiol (Mountant, Permafluor, Thermo Fisher Scientific, UK) and examined under a confocal microscope (TCS SP8, Leica Microsystems, Rueil-Malmaison, France) equipped with an oil objective (Type F immersion liquid, ne 23 = 1,5180, ve = 46, Leica Microsystems). Data were acquired with LASAF software, version 1.62 (Leica Microsystems). Mouse lungs were embedded in a cryogenic gel and serial longitudinal cryosections were stained and observed as previously reported 13 .Scientific RepoRtS | (2020) 10:914 | https://doi.
In vitro, the differentiation of megakaryocytes (MKs) is improved by aryl-hydrocarbon receptor (AHR) antagonists such as StemRegenin 1 (SR1), an effect physiologically recapitulated by the presence of stromal mesenchymal cells (MSC). This inhibition promotes the amplification of a CD34+CD41low population able to mature as MKs with a high capacity for platelet production. In this short report, we showed that the emergence of the thrombocytogenic precursors and the enhancement of platelet production triggered by SR1 involved Ikaros. The downregulation/inhibition of Ikaros (shRNA or lenalidomide) significantly reduced the emergence of SR1-induced thrombocytogenic population, suggesting a crosstalk between AHR and Ikaros. Interestingly, using a proximity ligation assay, we could demonstrate a physical interaction between AHR and Ikaros. This interaction was also observed in the megakaryocytic cells differentiated in the presence of MSCs. In conclusion, our study revealed a previously unknown AHR/ Ikaros -dependent pathway which prompted the expansion of the thrombocytogenic precursors. This AHR- Ikaros dependent checkpoint controlling MK maturation opens new perspectives to platelet production engineering.
Human CD34+ progenitors can be differentiated in vitro into proplatelet-producing megakaryocytes (MKs) within 17 days. During this time, four cell populations emerge, phenotypically defined as CD34+CD41+ on day 7 (D7) and CD34+CD41+CD9- on D10 and D14 - qualified as productive because they can differentiate into proplatelet-forming cells during the D14-D17 period - and CD34-CD41+ or CD34+CD41+CD9+ on day 10 - qualified as unproductive because they are unable to form proplatelets later. Coculture with mesenchymal stem cells, or the presence of the AHR antagonist SR1, boosts the productive pathway in two ways: firstly, it increases the yield of D10 and D14 CD34+CD41+CD9- cells and secondly, it greatly increases their ability to generate proplatelets; in contrast, SR1 has no noticeable effect on the unproductive cell types. A transcriptome analysis was performed to decipher the genetic basis of these properties. This work represents the first extensive description of the genetic perturbations which accompany the differentiation of CD34+ progenitors into mature MKs at a subpopulation level. It highlights a wide variety of biological changes modulated in a time-dependent manner and allows anyone, according to his/her interests, to focus on specific biological processes accompanying MK differentiation. For example, the modulation of the expression of genes associated with cell proliferation, lipid and cholesterol synthesis, extracellular matrix components, intercellular interacting receptors and MK and platelet functions reflected the chronological development of the productive cells and pointed to unsuspected pathways. Surprisingly, SR1 only affected the gene expression profile of D10 CD34+CD41+CD9- cells; thus, as compared to these cells and those present on D14, the poorly productive D10 CD34+CD41+CD9- cells obtained in the absence of SR1 and the two unproductive populations present on D10 displayed an intermediate gene expression pattern. In other words, the ability to generate proplatelets between D10 and D14 appeared to be linked to the capacity of SR1 to delay MK differentiation, meanwhile avoiding intermediate and inappropriate genetic perturbations. Paradoxically, the D14 CD34+CD41+CD9- cells obtained under SR1- or SR1+ conditions were virtually identical, raising the question as to whether their strong differences in terms of proplatelet production, in the absence of SR1 and between D14 and D17, are mediated by miRNAs or by memory post-translational regulatory mechanisms.
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