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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 slightly delays the maturation 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 with cKit, CD41 and CD42b fluorescent antibodies. A: 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 (CD41HighCD42bHigh). Note that most cells in gate 8 do not express c-Kit supporting our interpretation that they correspond to the most mature megakaryocytes. B: Histograms showing relative proportions of most mature CD41HighCD42bHigh 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. C: Histograms showing the absence of significant variations in the relative MFIs of CD41 (black bars) and CD42b (white bars) among CD41HighCD42bHigh cells.
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pone.0153860.g005: Culture on rDLL1 slightly delays the maturation 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 with cKit, CD41 and CD42b fluorescent antibodies. A: 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 (CD41HighCD42bHigh). Note that most cells in gate 8 do not express c-Kit supporting our interpretation that they correspond to the most mature megakaryocytes. B: Histograms showing relative proportions of most mature CD41HighCD42bHigh 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. C: Histograms showing the absence of significant variations in the relative MFIs of CD41 (black bars) and CD42b (white bars) among CD41HighCD42bHigh cells.

Mentions: In a complementary approach, we used flow cytometry analyses to compare the maturation and polyploidization of megakaryocytes generated by CD9High MEPs after a 5 days culture on either IgGs or rDLL1. Semi-quantitative analyses revealed that CD9High MEPs generated an increased number of viable cells (mainly Kit+ progenitor cells) on rDLL1 than on IgGs (S6 Fig) but the proportion of maturing CD41+/CD42b+ double positive megakaryocytic cells did not change significantly (data not shown). However, we found that the culture on rDLL1 induced a slight but significant (p = 0.01 in Student t-test) decrease in the proportion of CD41+/CD42b+ cells displaying the highest levels of both CD41 and CD42b (CD41HighCD42bHigh gate 8, Fig 5A and 5B) when compared to other control conditions. Interestingly, the decrease in the proportion of these CD41HighCD42bHigh megakaryocytes (most of which did not express c-kit; Fig 5A) was attenuated among Kit-CD41+CD42b+ (Fig 5B; compare black and white bars) and their mean expression levels of CD41 and CD42b (Fig 5C) did not change significantly thus suggesting a delay rather than strong inhibition of terminal megakaryocytic differentiation.


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 slightly delays the maturation 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 with cKit, CD41 and CD42b fluorescent antibodies. A: 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 (CD41HighCD42bHigh). Note that most cells in gate 8 do not express c-Kit supporting our interpretation that they correspond to the most mature megakaryocytes. B: Histograms showing relative proportions of most mature CD41HighCD42bHigh 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. C: Histograms showing the absence of significant variations in the relative MFIs of CD41 (black bars) and CD42b (white bars) among CD41HighCD42bHigh cells.
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Related In: Results  -  Collection

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pone.0153860.g005: Culture on rDLL1 slightly delays the maturation 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 with cKit, CD41 and CD42b fluorescent antibodies. A: 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 (CD41HighCD42bHigh). Note that most cells in gate 8 do not express c-Kit supporting our interpretation that they correspond to the most mature megakaryocytes. B: Histograms showing relative proportions of most mature CD41HighCD42bHigh 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. C: Histograms showing the absence of significant variations in the relative MFIs of CD41 (black bars) and CD42b (white bars) among CD41HighCD42bHigh cells.
Mentions: In a complementary approach, we used flow cytometry analyses to compare the maturation and polyploidization of megakaryocytes generated by CD9High MEPs after a 5 days culture on either IgGs or rDLL1. Semi-quantitative analyses revealed that CD9High MEPs generated an increased number of viable cells (mainly Kit+ progenitor cells) on rDLL1 than on IgGs (S6 Fig) but the proportion of maturing CD41+/CD42b+ double positive megakaryocytic cells did not change significantly (data not shown). However, we found that the culture on rDLL1 induced a slight but significant (p = 0.01 in Student t-test) decrease in the proportion of CD41+/CD42b+ cells displaying the highest levels of both CD41 and CD42b (CD41HighCD42bHigh gate 8, Fig 5A and 5B) when compared to other control conditions. Interestingly, the decrease in the proportion of these CD41HighCD42bHigh megakaryocytes (most of which did not express c-kit; Fig 5A) was attenuated among Kit-CD41+CD42b+ (Fig 5B; compare black and white bars) and their mean expression levels of CD41 and CD42b (Fig 5C) did not change significantly thus suggesting a delay rather than strong inhibition of terminal megakaryocytic differentiation.

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