Extracellular vesicles synchronize cellular phenotypes of differentiating cells

During embryonic development, cells differentiate in a coordinated manner, aligning their fate decisions and differentiation stages with those of surrounding cells. However, little is known about the mechanisms that regulate this synchrony. Here, Kyoto University researchers show that cells in close proximity synchronize their differentiation stages and cellular phenotypes with each other via extracellular vesicle (EV)-mediated cellular communication. The researchers previously established a mouse embryonic stem cell (ESC) line harbouring an inducible constitutively active protein kinase A (CA-PKA) gene and found that the ESCs rapidly differentiated into mesoderm after PKA activation. In the present study, they performed a co-culture of Control-ESCs and PKA-ESCs, finding that both ESC types rapidly differentiated in synchrony even when PKA was activated only in PKA-ESCs, a phenomenon they named ‘Phenotypic Synchrony of Cells (PSyC)’. The researchers further demonstrated PSyC was mediated by EVs containing miR-132. PKA-ESC-derived EVs and miR-132-containing artificial nano-vesicles similarly enhanced mesoderm and cardiomyocyte differentiation in ESCs and ex vivo embryos, respectively. PSyC is a new form of cell-cell communication mediated by the EV regulation of neighbouring cells and could be broadly involved in tissue development and homeostasis.

PSyC is mediated by EVs

(a) Schematic diagram of the chimeric aggregate co-culture differentiation system with EV secretion inhibited. PKA-ESCs and Control-ESCs were seeded in low adhesion dishes at a 3:1 ratio to create chimeric aggregates, and differentiation induction was initiated by the depletion of LIF. 24 h later, the aggregates were replated on normal plates and cultured with EV secretion inhibitors. (b) The number of Flk1⁺ cells was reduced with treatment of the EV secretion inhibitors manumycin A (10 μM) or GW4869 (5 μM). PKA-ESC and Control-ESC chimeric aggregates on D3.5 under Dox- condition were analysed by FACS. (c) Percentage of PKA-ESCs and Control-ESCs in chimeric aggregates expressing Flk1 under Dox- condition. Data were analysed using the Kruskal-Wallis test followed by the Steel-Dwass test. (d) Immunostaining for Flk1⁺ Control-ESCs (white) in chimeric aggregates with manumycin A (10 μM), GW4869 (5 μM), or DMSO (control). (e) Schematic diagram of the EV collection and treatment. Control-ESCs aggregates were plated without LIF. EVs were collected from the conditioned medium of PKA-ESCs as pellets and added to Control-ESCs aggregates. (f) Immunoblots of EV markers CD81 and Flotillin-1, non-EV marker calnexin in the cell or EV lysate (30 μg for CD81 and calnexin, 3 μg for Flotillin-1). (g) Transmission electron microscopy image of EVs from the conditioned medium. (h) Analysis of the EV size distribution in Dox+ (left) and Dox- (right) conditioned medium. (i) FACS analysis of Flk1⁺ cells in EV-treated Control-ESCs on D3.5. (j) Percentage of Control-ESCs expressing Flk1. Data were analysed using the Kruskal-Wallis test followed by the Steel-Dwass test. (k) Immunostaining for mesodermal markers (Flk1, PDGFRα) in EV-treated Control-ESCs on D3.5