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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

Culture on rDLL1 decreases the ploidy of megakaryocytes generated by CD9High MEPs.Equal numbers of CD9High 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 for CD41, CD42b, c-Kit and DNA content. Histograms show the percentages of the different classes of polyploidy of Kit-/CD41+/CD42b+ cells generated on IgGs (full boxes) or rDLL1 (hatched boxes) in the absence (left panel) or presence (right panel) of DAPT (Means and standard deviations from 3 independent experiments). Statistically significant variations between conditions (p < 0.05 in Student t-test) are indicated by asterisks (NS: non significant variation).
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pone.0153860.g006: Culture on rDLL1 decreases the ploidy of megakaryocytes generated by CD9High MEPs.Equal numbers of CD9High 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 for CD41, CD42b, c-Kit and DNA content. Histograms show the percentages of the different classes of polyploidy of Kit-/CD41+/CD42b+ cells generated on IgGs (full boxes) or rDLL1 (hatched boxes) in the absence (left panel) or presence (right panel) of DAPT (Means and standard deviations from 3 independent experiments). Statistically significant variations between conditions (p < 0.05 in Student t-test) are indicated by asterisks (NS: non significant variation).

Mentions: Polyploidization of megakaryocytes generated in the same culture conditions was addressed by FACS analyses after DNA labeling. We found a 2 fold decrease in the mean percentage of polyploid Kit-/CD41+/CD42b+ cells generated on rDLL1 as compared with IgGs (7 ± 6% of ≥ 8N on rDLL1 vs 15 ± 5% on IgGs) with a significant decrease in the percentage of 16 N cells (Fig 6A). However, the percentage of 4N Kit-/CD41+/CD42b+ cells (including cells in S phase) generated on rDLL1 significantly increased as compared with IgGs (Fig 6A) thus suggesting concomitant increase of cycling cells as also suggested by cell cycle analyses performed at day 2 (S7 Fig). Importantly, none of these changes were detected in the presence of DAPT (Fig 6B).


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)

Culture on rDLL1 decreases the ploidy of megakaryocytes generated by CD9High MEPs.Equal numbers of CD9High 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 for CD41, CD42b, c-Kit and DNA content. Histograms show the percentages of the different classes of polyploidy of Kit-/CD41+/CD42b+ cells generated on IgGs (full boxes) or rDLL1 (hatched boxes) in the absence (left panel) or presence (right panel) of DAPT (Means and standard deviations from 3 independent experiments). Statistically significant variations between conditions (p < 0.05 in Student t-test) are indicated by asterisks (NS: non significant variation).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153860.g006: Culture on rDLL1 decreases the ploidy of megakaryocytes generated by CD9High MEPs.Equal numbers of CD9High 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 for CD41, CD42b, c-Kit and DNA content. Histograms show the percentages of the different classes of polyploidy of Kit-/CD41+/CD42b+ cells generated on IgGs (full boxes) or rDLL1 (hatched boxes) in the absence (left panel) or presence (right panel) of DAPT (Means and standard deviations from 3 independent experiments). Statistically significant variations between conditions (p < 0.05 in Student t-test) are indicated by asterisks (NS: non significant variation).
Mentions: Polyploidization of megakaryocytes generated in the same culture conditions was addressed by FACS analyses after DNA labeling. We found a 2 fold decrease in the mean percentage of polyploid Kit-/CD41+/CD42b+ cells generated on rDLL1 as compared with IgGs (7 ± 6% of ≥ 8N on rDLL1 vs 15 ± 5% on IgGs) with a significant decrease in the percentage of 16 N cells (Fig 6A). However, the percentage of 4N Kit-/CD41+/CD42b+ cells (including cells in S phase) generated on rDLL1 significantly increased as compared with IgGs (Fig 6A) thus suggesting concomitant increase of cycling cells as also suggested by cell cycle analyses performed at day 2 (S7 Fig). Importantly, none of these changes were detected in the presence of DAPT (Fig 6B).

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