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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 recombinant Notch ligand rDLL1 stimulates the amplification of bipotent and erythroid 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) in plate culture wells coated with either control IgG1 or recombinant rDLL1 in the presence or absence of DAPT as indicated. Total numbers of the 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 different types of colonies generated from untreated cells (Day 0) and after a two days culture on IgG, rDLL1 or rDLL1 + DAPT. Right histograms shows 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 histogram. 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.g002: MEP cells culture on recombinant Notch ligand rDLL1 stimulates the amplification of bipotent and erythroid 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) in plate culture wells coated with either control IgG1 or recombinant rDLL1 in the presence or absence of DAPT as indicated. Total numbers of the 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 different types of colonies generated from untreated cells (Day 0) and after a two days culture on IgG, rDLL1 or rDLL1 + DAPT. Right histograms shows 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 histogram. 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: In a second series of experiments, sorted MEPs were cultured for 2 days either on coated IgGs or on coated recombinant rDLL1 Notch ligand in the presence or absence of γ-secretase inhibitor DAPT. Again, when compared with Day 0, the 2 days culture of MEPs on rDLL1 significantly increased the total number of colonies (Fig 2A) as well as the number of mixed colonies (Fig 2C) while these changes were reversed by DAPT and were not observed after culture on control IgGs. In the same conditions but in contrast to co-cultures with OP9-DLL1 cells, cultures on rDLL1 significantly decreased the number of megakaryocytic colonies (Fig 2D) and significantly increased the number of erythroid colonies (Fig 2B). Interestingly, the net increase in erythroid colonies (mean increase of 350 colonies; Fig 2B left panel) largely exceeded the small decrease in megakaryocytic colonies (mean decrease of 50 colonies; Fig 2D left panel) thus suggesting the selective amplification of committed erythroid progenitors. Importantly, all changes observed on rDLL1 were reversed by DAPT and were not observed in cultures performed on IgGs. Moreover, qRT-PCR analyses performed at day 2 confirmed the expected increase in Hes1 transcripts consistent with Notch pathway activation in the presence of rDLL1 but not on IgGs and its partial repression by DAPT (S2 Fig). In complementary experiments, we determined that the increase of mixed and erythroid colonies induced by rDLL1 remained strictly dependent on the presence of SCF (S3 Fig) and associated with increased expression of c-Kit (S2 Fig).


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 recombinant Notch ligand rDLL1 stimulates the amplification of bipotent and erythroid 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) in plate culture wells coated with either control IgG1 or recombinant rDLL1 in the presence or absence of DAPT as indicated. Total numbers of the 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 different types of colonies generated from untreated cells (Day 0) and after a two days culture on IgG, rDLL1 or rDLL1 + DAPT. Right histograms shows 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 histogram. 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

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

pone.0153860.g002: MEP cells culture on recombinant Notch ligand rDLL1 stimulates the amplification of bipotent and erythroid 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) in plate culture wells coated with either control IgG1 or recombinant rDLL1 in the presence or absence of DAPT as indicated. Total numbers of the 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 different types of colonies generated from untreated cells (Day 0) and after a two days culture on IgG, rDLL1 or rDLL1 + DAPT. Right histograms shows 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 histogram. 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: In a second series of experiments, sorted MEPs were cultured for 2 days either on coated IgGs or on coated recombinant rDLL1 Notch ligand in the presence or absence of γ-secretase inhibitor DAPT. Again, when compared with Day 0, the 2 days culture of MEPs on rDLL1 significantly increased the total number of colonies (Fig 2A) as well as the number of mixed colonies (Fig 2C) while these changes were reversed by DAPT and were not observed after culture on control IgGs. In the same conditions but in contrast to co-cultures with OP9-DLL1 cells, cultures on rDLL1 significantly decreased the number of megakaryocytic colonies (Fig 2D) and significantly increased the number of erythroid colonies (Fig 2B). Interestingly, the net increase in erythroid colonies (mean increase of 350 colonies; Fig 2B left panel) largely exceeded the small decrease in megakaryocytic colonies (mean decrease of 50 colonies; Fig 2D left panel) thus suggesting the selective amplification of committed erythroid progenitors. Importantly, all changes observed on rDLL1 were reversed by DAPT and were not observed in cultures performed on IgGs. Moreover, qRT-PCR analyses performed at day 2 confirmed the expected increase in Hes1 transcripts consistent with Notch pathway activation in the presence of rDLL1 but not on IgGs and its partial repression by DAPT (S2 Fig). In complementary experiments, we determined that the increase of mixed and erythroid colonies induced by rDLL1 remained strictly dependent on the presence of SCF (S3 Fig) and associated with increased expression of c-Kit (S2 Fig).

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