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Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells
Published on: November 1, 2017
DLGAP1 directs megakaryocytic growth and differentiation in an MPL dependent manner in hematopoietic cells
Boguslaw A Kwiatkowski1, Nicolas R Burwick1, Robert E Richard1
1Seattle Institute for Biomedical and Clinical Research, VA Puget Sound Healthcare System, 1660 South Columbian Way, S-111-ONC, Seattle, WA 98108 USA.
Background:
The MPL protein is a major regulator of megakaryopoiesis and platelet formation as well as stem cell regulation. Aberrant MPL and downstream Jak/STAT signaling results in the development of the Myeloproliferative Neoplasms (MPN). The pathogenetic and phenotypic features of the classical MPNs cannot be explained by the known mutations and genetic variants associated with the disease.
Methods:
In order to identify potential pathways involved in MPN development, we have performed a functional screen using retroviral insertional mutagenesis in cells dependent on MPL activation. We have used viral transduction and plasmid transfections to test the effects of candidate gene overexpression on growth and differentiation of megakaryocytic cells. The shRNA approach was used to test for the effects of candidate gene downregulation in cells. All effects were tested with candidate gene alone or in presence of hematopoietic relevant kinases in the growth medium. We assayed the candidate gene cellular localization in varying growth conditions by immunofluorescence. Flow Cytometry was used for testing of transduction efficiency and for sorting of positive cells.
Results:
We have identified the DLGAP1 gene, a member of the Scribble cell polarity complex, as one of the most prominent positive candidates. Analyses in hematopoietic cell lines revealed DLGAP1 centrosomal and cytoplasmic localization. The centrosomal localization of DLGAP1 was cell cycle dependent and hematopoietic relevant tyrosine kinases: Jak2, SRC and MAPK as well as the CDK1 kinase promoted DLGAP1 dissociation from centrosomes. DLGAP1 negatively affected the growth rate of MPL dependent hematopoietic cells and supported megakaryocytic cells polyploidization, which was correlated with its dissociation from centrosomes.
Conclusions:
Our data support the conclusion that DLGAP1 is a novel, potent factor in MPL signaling, affecting megakaryocytic growth and differentiation, relevant to be investigated further as a prominent candidate in MPN development.
Insights
The DLGAP1 gene negatively impacts hematopoietic cell growth and aids megakaryocyte polyploidization, suggesting its role in Myeloproliferative Neoplasms (MPN) development.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- MPL protein is crucial for megakaryopoiesis, platelet formation, and stem cell regulation.
- Aberrant MPL signaling and Jak/STAT pathway activation are implicated in Myeloproliferative Neoplasms (MPN).
- Known mutations do not fully explain MPN pathogenesis and phenotypes.
Purpose of the Study:
- To identify novel pathways involved in MPN development.
- To investigate the role of candidate genes in MPL-dependent cell growth and differentiation.
Main Methods:
- Functional screening using retroviral insertional mutagenesis in MPL-dependent cells.
- Overexpression and shRNA approaches to assess candidate gene effects.
- Immunofluorescence and Flow Cytometry for cellular localization and cell sorting.
Main Results:
- DLGAP1, a component of the Scribble cell polarity complex, was identified as a key candidate.
- DLGAP1 exhibits cell cycle-dependent centrosomal and cytoplasmic localization.
- Hematopoietic kinases (Jak2, SRC, MAPK, CDK1) promote DLGAP1 dissociation from centrosomes.
- DLGAP1 negatively regulates MPL-dependent cell growth and promotes megakaryocyte polyploidization.
Conclusions:
- DLGAP1 is a novel factor in MPL signaling, influencing megakaryocytic growth and differentiation.
- DLGAP1's role in megakaryopoiesis warrants further investigation for MPN development.
- DLGAP1 is a potential therapeutic target for MPN.
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