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MiRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate.

ABSTRACT: When stem cells and multipotent progenitors differentiate, they undergo fate restriction, enabling a single fate and blocking differentiation along alternative routes. We herein present a mechanism whereby such unequivocal commitment is achieved, based on microRNA (miRNA)-dependent repression of an alternative cell fate. We show that the commitment of monocyte RAW264.7 progenitors to active macrophage differentiation involves rapid up-regulation of miR-155 expression, which leads to the suppression of the alternative pathway, namely RANK ligand-induced osteoclastogenesis, by repressing the expression of MITF, a transcription factor essential for osteoclast differentiation. A temporal asymmetry, whereby miR-155 expression precedes and overrides the activation of the osteoclast transcriptional program, provides the means for coherent macrophage differentiation, even in the presence of osteoclastogenic signals. Based on these findings, we propose that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.

SUBMITTER: Mann M

PROVIDER: S-EPMC2936650 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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miRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate.

Mann Mati M Barad Omer O Agami Reuven R Geiger Benjamin B Hornstein Eran E

Proceedings of the National Academy of Sciences of the United States of America 20100818 36

When stem cells and multipotent progenitors differentiate, they undergo fate restriction, enabling a single fate and blocking differentiation along alternative routes. We herein present a mechanism whereby such unequivocal commitment is achieved, based on microRNA (miRNA)-dependent repression of an alternative cell fate. We show that the commitment of monocyte RAW264.7 progenitors to active macrophage differentiation involves rapid up-regulation of miR-155 expression, which leads to the suppress ...[more]

PMID: 20720163

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Project description:Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Project description:BackgroundImproving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis.MethodsEffects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET).ResultsIn contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation.ConclusionsOur findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

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MiRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate.

Publications

miRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate.

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