Extracellular vesicles (EVs) are important mediators of intercellular communication and have a broad range of regulatory roles in physiology and disease. In Developmental Cell, Cerione and colleagues now find that embryonic stem cells (ESCs) produce EVs that promote the maintenance of the pluripotent state.

The authors first established a mouse ESC line that expressed key pluripotency-associated proteins (Oct3/4, Nanog, Sox2, Myc and Klf4) and satisfied two assays to test for pluripotency, namely spheroid formation and alkaline phosphatase activity. When analysing the ESC-conditioned culture medium with nanoparticle tracking analysis, they found that the two major types of EVs, exosomes and larger microvesicles, were both present. Moreover, these ESC-generated EVs were capable of binding to the surface of other ESCs. Importantly, when ESCs were transferred to a medium that promotes differentiation, several differentiation-associated characteristics were suppressed. For example, differentiating cells have substantially lower proliferation rates, stop expressing Oct3/4 and Nanog, accumulate the epigenetic mark trimethylation of lysine 27 on histone H3 (H3K27me3) and lose alkaline phosphatase activity and the ability to form spheroids. All such features were reversed when adding ESC-derived EVs.

EVs promote stemness

Credit: V. Summersby/Springer Nature Limited

So how do EVs promote pluripotent stem cell features? The authors found that adding ESC-derived EVs to the culture medium that promotes differentiation led to the strong phosphorylation-dependent activation of focal adhesion kinase (FAK) and that treating the cells with a FAK inhibitor was sufficient to induce the loss of pluripotency markers. Moreover, ectopic expression of an activated form of FAK in ESCs grown in a differentiation medium enabled cells to maintain the phenotype of pluripotent stem cells. Thus, EV-dependent FAK activation is important for the maintenance of pluripotency in cultured ESCs. Furthermore, ESC-derived EVs were rich in fibronectin, which was found to engage with recipient cell integrins and activate FAK. Indeed, treatment with a peptide that competes with fibronectin for the binding to integrins abolished the effect of fibronectin.

“EV-dependent FAK activation is important for the maintenance of pluripotency in cultured ESCs”

But what is the in vivo relevance of such findings? The pluripotent cell state that is maintained in culture in the form of ESCs is found only transiently in the epiblast of blastocyst-stage embryos, before such epiblast cells start differentiating into the germline, ectoderm, mesoderm and endoderm — to eventually produce all cells that make up an organism. When injected in mouse blastocysts, EV-treated cells successfully incorporated into the embryos, giving rise to chimeric mice, indicating that the cells are pluripotent. Thus, the authors suggest EVs might be produced by pluripotent cells to stabilize the pluripotent state and inhibit premature differentiation during embryo development. However, such role in mice remains to be confirmed.

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Correspondence to Kim Baumann.

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EVs promote stemness

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Baumann, K. EVs promote stemness. Nat Rev Mol Cell Biol (2021). https://doi.org/10.1038/s41580-020-00327-5

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