Comparison of the Notch response of sorted MEP cells expressing different levels of CD9.

<p>Progeny analyses of equal numbers of sorted CD9^Med and CD9^High MEP subsets were performed by colony assays before (Day 0) and after a two days culture either on IgG or rDLL1 with or without DAPT as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153860#pone.0153860.g002" target="_blank">Fig 2</a>. The numbers of different types of colonies generated by either CD9^Med or CD9^High MEP are presented on left (A, B, C, D, E) and right (F, G, H, I, J) histograms respectively (means and standard deviations from 3 independent MEP preparations). Results of ANOVA analyses performed on each dataset are indicated above each corresponding histogram. <b>A, B:</b> Piled histograms showing the cumulated numbers of the different types of colonies generated by CD9^Med (A) or CD9^High MEP (F). Results of ANOVA analysis performed on the total numbers of colonies are indicated above the histogram. <b>B, G:</b> Histograms showing the numbers of erythroid colonies generated by CD9^Med (B) or CD9^High MEP (G). <b>C, H:</b> Histograms showing the numbers of mixed colonies generated by CD9^Med (C) or CD9^High MEP (H). <b>D, I:</b> Histograms showing the numbers of megakaryocytic colonies generated by CD9^Med (D) or CD9^High MEP (I). <b>E, J:</b> Histograms showing the numbers of myeloid colonies generated by CD9^Med (E) or CD9^High MEP (J). Statistically significant differences between conditions validated by either ANOVA analyses followed by Tukey’s test or by Student t-test are indicated by full and dotted braces respectively associated with corresponding p values.</p

Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9^High Committed Megakaryocytic Progenitors

<div><p>This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit⁺ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9^High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9^High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9^High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.</p></div

Culture on rDLL1 slightly delays the maturation of megakaryocytes generated by CD9^High MEPs.

<p>Equal numbers of CD9^High MEP cells were cultured for 5 days in the presence of a complete cocktail of myeloid cytokines (IL3, SCF, EPO, GM-CSF, TPO, Flt3L, IL11) in plate culture wells coated with either control IgG1 or recombinant rDLL1 in the presence or absence of DAPT followed by FACS analyses after labeling with cKit, CD41 and CD42b fluorescent antibodies. <b>A:</b> FACS dot-plots showing the expression of CD41 and CD42b with red and blue dots corresponding to cKit⁺ and cKit^- cells respectively. Gate P8 is defined by CD41⁺CD42b⁺ double positive cells displaying the highest levels of both CD41 and CD42b (CD41^HighCD42b^High). Note that most cells in gate 8 do not express c-Kit supporting our interpretation that they correspond to the most mature megakaryocytes. <b>B:</b> Histograms showing relative proportions of most mature CD41^HighCD42b^High megakaryocytes (gate 8) among all CD41⁺CD42b⁺ cells (gate Q2) including (black bars) or not (white bars) Kit⁺ cells (Means and standard deviations from 3 independent experiments). Statistically significant variations are indicated by braces with corresponding p-values in Student t-test. <b>C:</b> Histograms showing the absence of significant variations in the relative MFIs of CD41 (black bars) and CD42b (white bars) among CD41^HighCD42b^High cells.</p

Single cell progeny analyses of CD9^Med and CD9^High MEPs with or without Notch activation.

<p>Single CD9^Med or CD9^High MEP were individually seeded in 96 wells culture plates that have been coated with either IgGs or rDLL1 and containing medium supplemented with a complete cocktail of myeloid cytokines. The different types of developed colonies were numbered at day 7 by visual inspection under bright light microscope as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153860#pone.0153860.s008" target="_blank">S8 Fig</a>. <b>A, B</b>: Repartition of the indicated type of colony as a percentage of all single seeded CD9^Med MEP (<b>A</b>) and as a percentage of CD9^Med MEP raising colonies (<b>B</b>) (Means and standard deviations from 3 independent experiments). <b>C, D</b>: Repartition of the indicated type of colony as a percentage of all single seeded CD9^High MEP (<b>C</b>) and as a percentage of CD9^High MEP raising colonies (<b>D</b>) (Means and standard deviations from 3 independent experiments). Tables on the right display p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in Tables and by asterisks on right histograms.</p

MEP cells culture on OP9 stromal cells expressing Notch ligand DLL1 stimulates the amplification of bipotent progenitors.

<p>2000 bone marrow MEP cells were cultured for two days in the presence of a complete cocktail of myeloid cytokines (IL3, SCF, EPO, GM-CSF, TPO, Flt3L, IL11) either on control OP9 or on OP9-DLL1 stromal cells expressing Notch ligand DLL1 in the presence or absence of γ-secretase inhibitor DAPT as indicated. Total numbers of bipotent erythro-megakaryocytic (Ery/Meg), pure erythroid (Ery) or megakaryocytic (Meg) progenitors present in the initial population (day 0) and after the two days culture in the different conditions were determined by colony assays performed in semi-solid medium in the presence of the same complete cocktail of cytokines. Absolute and relative numbers of different types of colonies (normalized to that obtained on day 0) are presented on left and right histograms respectively (means and standard deviations from 5 independent MEP preparations). <b>A</b>: Left panel shows piled histograms of the numbers of erythroid (Ery), megakaryocytic (Meg) and mixed (Ery/meg) colonies generated from untreated cells (Day 0) and after a two days culture on OP9, OP9-DLL1 or OP9-DLL1 stromal cells + DAPT. Right histograms show the relative total numbers of colonies. <b>B, C, D:</b> Histograms showing separately the numbers (left panels) and relative numbers (right panels) of erythroid (<b>B</b>), mixed (<b>C</b>) and megakaryocytic (<b>D</b>) colonies obtained in the different culture conditions (same data as in <b>A</b>). Results of ANOVA analyses performed on each dataset are indicated above each corresponding histograms. Statistically significant differences between conditions are indicated by braces with corresponding post-hoc p-values for the Tukey’s test indicated in bold characters. Statistically significant differences validated in Student t-test only are indicated by dotted braces.</p

Culture on rDLL1 decreases the number of single megakaryocytes and increases the size of megakaryocytic colonies generated by CD9^High MEPs.

<p>CD9^High MEPs were cultured for two days on either IgGs or rDLL1 before analysis of their progeny by colony assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153860#pone.0153860.g003" target="_blank">Fig 3</a> except that single megakaryocytes and megakaryocytic colonies displaying different numbers of megakaryocytes were numbered separately. <b>A:</b> Piled histograms showing the numbers (left panel) and percentages (right panel) of single megakaryocytes (MK1), megakaryocytic colonies containing at least 2 megakaryocytes (MK ≥2) as well as few erythroid (Ery), myeloid (Myelo) and mixed erythro-megakaryocytic (Ery/Mk) colonies generated after the 2 days culture on either IgGs or rDLL1 (mean and standard deviations from 3 independent MEP preparations). Table on the right displays p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in table and by asterisks on the right histogram. <b>B:</b> Histograms showing the percentages of different sizes of pure megakaryocytic colonies (from single to 8 and more megakaryocytes) on IgGs or rDLL1 (means and standard deviations from the same 3 independent MEP preparations as in A). Table on the right displays p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in Table and by full braces and asterisks on histogram.</p