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Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia
Aya Abou Hammoud1,2,3, Nina Kirstein1,2, Virginie Mournetas1,2
1Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France.
Abstract:
Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF-mediated reprogramming are poorly understood. By employing a LIF-dependent 'plasticity' test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O2 despite lower levels of Pluri and Master gene expression in comparison to 20% O2.
Insights
Klf5 is a key regulator of mouse embryonic stem cell (mESC) plasticity, influencing differentiation and pluripotency. PI3K signaling impacts committed cells, while MMP1 partially mimics leukemia inhibitory factor (LIF) effects.
Area of Science:
- Stem cell biology
- Developmental biology
- Molecular signaling
Background:
- Mouse embryonic stem cells (mESCs) require leukemia inhibitory factor (LIF) for pluripotency maintenance.
- LIF withdrawal induces differentiation, with distinct reversible and irreversible phases.
- Regulators of LIF-mediated reprogramming and mESC plasticity remain poorly understood.
Purpose of the Study:
- To identify key effectors of LIF in mESC plasticity.
- To investigate the role of PI3K signaling in mESC pluripotency and differentiation.
- To assess the effect of MMP1 and oxygen levels on mESC plasticity.
Main Methods:
- Development of a LIF-dependent 'plasticity' test.
- Analysis of Klf5 and JunB roles in mESC differentiation.
- Assessment of PI3K signaling pathway activity.
- Evaluation of MMP1 and hypoxic conditions (3% O2) on mESC pluripotency and gene expression.
Main Results:
- Klf5, not JunB, was identified as a key effector of LIF in mESC plasticity.
- PI3K signaling is crucial for committed cells, promoting mesodermal markers (Brachyury) over endoderm/neuroectoderm.
- MMP1 partially mimicked LIF's effect on plasticity, but less efficiently.
- mESCs retained plasticity and pluripotency under hypoxia (3% O2), despite altered Pluri/Master gene expression.
Conclusions:
- Klf5 is a critical regulator of mESC plasticity and LIF-mediated reprogramming.
- PI3K signaling differentially regulates pluripotency and lineage commitment.
- MMP1 offers partial functional redundancy to LIF.
- Hypoxia does not abrogate mESC plasticity and pluripotency in vitro.
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