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Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

Weiss-Gayet M, Starck J, Chaabouni A, Chazaud B, Morlé F - PLoS ONE (2016)

Bottom Line: 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.Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High 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.

View Article: PubMed Central - PubMed

Affiliation: Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon1, Villeurbanne, France.

ABSTRACT
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 CD9High 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 CD9High 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 CD9High 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.

No MeSH data available.


Related in: MedlinePlus

MEP cells culture on OP9 stromal cells expressing Notch ligand DLL1 stimulates the amplification of bipotent progenitors.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). A: 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, C, D: Histograms showing separately the numbers (left panels) and relative numbers (right panels) of erythroid (B), mixed (C) and megakaryocytic (D) colonies obtained in the different culture conditions (same data as in A). 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.
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pone.0153860.g001: MEP cells culture on OP9 stromal cells expressing Notch ligand DLL1 stimulates the amplification of bipotent progenitors.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). A: 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, C, D: Histograms showing separately the numbers (left panels) and relative numbers (right panels) of erythroid (B), mixed (C) and megakaryocytic (D) colonies obtained in the different culture conditions (same data as in A). 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.

Mentions: The first aim of our study was to determine the fate of erythro-megakaryocytic bipotent progenitors upon stimulation of the Notch pathway. For that purpose, we used colony assays to quantify the number of different progenitors present in sorted mouse bone marrow MEP population (lin-/Sca1-/Kit+/CD16/32low/CD34low; S1 Fig) before and after a 2 days stimulation of the Notch pathway in the presence of a complete cocktail of myeloid cytokines (IL3, Ftl3l, GM-CSF, SCF, IL11, EPO, TPO). 25% of freshly sorted MEPs generated colonies including 50% of pure erythroid colonies, 25% of pure megakaryocytic and 25% of mixed erythro-megakaryocytic colonies (Fig 1A, Day 0). These results confirmed previous studies, that the MEP population is actually composed of a mixture of pure erythroid and megakaryocytic progenitors in addition to bipotent progenitors. We also confirmed that this MEP population expressed Notch2 receptor (FACS analysis not shown) as previously reported by others [36].


Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

Weiss-Gayet M, Starck J, Chaabouni A, Chazaud B, Morlé F - PLoS ONE (2016)

MEP cells culture on OP9 stromal cells expressing Notch ligand DLL1 stimulates the amplification of bipotent progenitors.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). A: 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, C, D: Histograms showing separately the numbers (left panels) and relative numbers (right panels) of erythroid (B), mixed (C) and megakaryocytic (D) colonies obtained in the different culture conditions (same data as in A). 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835090&req=5

pone.0153860.g001: MEP cells culture on OP9 stromal cells expressing Notch ligand DLL1 stimulates the amplification of bipotent progenitors.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). A: 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, C, D: Histograms showing separately the numbers (left panels) and relative numbers (right panels) of erythroid (B), mixed (C) and megakaryocytic (D) colonies obtained in the different culture conditions (same data as in A). 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.
Mentions: The first aim of our study was to determine the fate of erythro-megakaryocytic bipotent progenitors upon stimulation of the Notch pathway. For that purpose, we used colony assays to quantify the number of different progenitors present in sorted mouse bone marrow MEP population (lin-/Sca1-/Kit+/CD16/32low/CD34low; S1 Fig) before and after a 2 days stimulation of the Notch pathway in the presence of a complete cocktail of myeloid cytokines (IL3, Ftl3l, GM-CSF, SCF, IL11, EPO, TPO). 25% of freshly sorted MEPs generated colonies including 50% of pure erythroid colonies, 25% of pure megakaryocytic and 25% of mixed erythro-megakaryocytic colonies (Fig 1A, Day 0). These results confirmed previous studies, that the MEP population is actually composed of a mixture of pure erythroid and megakaryocytic progenitors in addition to bipotent progenitors. We also confirmed that this MEP population expressed Notch2 receptor (FACS analysis not shown) as previously reported by others [36].

Bottom Line: 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.Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High 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.

View Article: PubMed Central - PubMed

Affiliation: Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon1, Villeurbanne, France.

ABSTRACT
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 CD9High 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 CD9High 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 CD9High 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.

No MeSH data available.


Related in: MedlinePlus