Astrocytes participate in synapse formation and neuron maturation during development and maintain brain homeostasis during adulthood. They are part of the blood-brain-barrier (BBB) which confers on the central nervous system a selective permeability that relies on transcellular mechanisms to transport molecules (Schiweck et al., 2018; Santello et al., 2019; Profaci et al., 2020). A key astrocyte feature is their ability to act as communication hubs by establishing astrocyte-neuron and astrocyte-astrocyte networks. These networks act via physical connections between astrocytic neuroligins and neuronal neurexins, through gap junctions between astrocytes, or by secreted factors that regulate synapse formation and maturation (Houades et al., 2008; Stogsdill et al., 2017; Patel and Weaver, 2021). Astrocyte released factors are implicated in neurodevelopmental disorders such as Rett Syndrome (RTT), where mutations in the transcriptional regulator methyl-CpG binding protein 2 (MECP2) induce defective neural networks (Ip et al., 2018; Tillotson and Bird, 2020). Despite neurons being the most affected cell type due to their high protein levels of MECP2, astrocytes also show an array of phenotypes (Albizzati et al., 2022; Sun et al., 2023). RTT astrocytes induce non-cell autonomous defects in neuron morphology and firing, thought to be driven by astrocyte released factors (Williams et al., 2014; Ehinger et al., 2021; Caldwell et al., 2022).

Astrocytes and neurons also communicate by secreting and internalizing extracellular vesicles (EVs). EVs are lipid-bilayer membrane-delimited vesicles generated in the cytoplasm or the multivesicular body (MVB)/ endosomal compartments, which contain cargo in the form of RNA, proteins and lipids (Yáñez-Mó et al., 2015; Welsh et al., 2024). EVs can regulate transcription and translation upon entering their target cells (Mulcahy et al., 2014). They are able to cross the BBB and have been detected in all biofluids using non-invasive methods relying on EV sedimentation by ultracentrifugation, precipitation using commercial kits, or size exclusion chromatography. In addition, EVs can be recovered from conditioned media in vitro (Antounians et al., 2019; O'Brien et al., 2020; Welsh et al., 2024). This feature has propelled the study of EV cargo for its potential as a diagnostic and prognostic tool to define RNA and proteins that are differentially expressed in disease (Lafourcade et al., 2016; Pistono et al., 2020; Upadhya et al., 2020). In the context of RTT, EVs isolated from mixed neuronal cultures can rescue RTT neuron networks, and their protein cargo was described as containing signaling proteins that are lacking in the absence of MECP2 (Sharma et al., 2019). In addition, Urine derived Stem Cells were shown to produce EVs with a miR-21-5p cargo that rescued neurogenesis and behaviour in a RTT mouse model (Pan et al., 2021). The potential for human astrocyte-derived EV (ADEV) cargo as a prognostic biomarker or as a therapy for neurodevelopmental disorders is unknown.

Sources of human astrocytes to study their ADEV cargo profile are limited. Primary astrocytes are isolated from human cadavers and astrocytes can be derived in vitro from human induced Pluripotent Stem Cells (iPSC). During development astrocytes emerge as a heterogeneous cell population arising from radial glia after neurogenesis is underway at week 24 post-conception in humans (Clarke and Barres, 2013). Generating astrocytes from iPSC has proven to be a similarly time-consuming endeavor, often requiring specialized equipment to generate a high yield of functionally mature astrocytic cultures (McCready et al., 2022). Recently, a novel differentiation approach has been shown to induce rapid differentiation into astrocytes that were validated by flow cytometry, bulk and single-cell RNASeq and shown to buffer extracellular glutamate, to integrate into the mouse brain after transplantation, and to support network activity of induced neurons in multi-electrode arrays (Lendemeijer et al., 2022). After generating neural progenitor cells (NPC) and enriching them by cell sorting, stimulation of the astrogliogenic JAK-STAT pathway with BMP4 and LIF generates functional human astrocyte cultures in just 28 days that support neuronal network activity (Lendemeijer et al., 2022). These functional human astrocyte cell cultures can be used to isolate ADEVs and characterize their cargo.

ADEVs contain micro-RNA (miRNA) cargo which affect the function of target pathways after internalization by the recipient cell (Lafourcade et al., 2016; Varcianna et al., 2019). In vitro experiments have shown that ADEVs are endocytosed by neurons, and their cargo influences neuronal transcripts and subsequently neuronal function (Venturini et al., 2019; Upadhya et al., 2020). In addition, ADEV cargo is altered depending on the reactive phenotype of the astrocytes, and is modified in response to cytokines, ATP or presence of pathogenic bacteria to incorporate miRNAs or proteins that regulate immune or neuronal responses (Casselli et al., 2017; Chaudhuri et al., 2018; You et al., 2020; Luarte et al., 2023). However, the basal miRNA cargo enrichment in ADEVs has not been studied in depth in healthy human astrocytes. Moreover, the use of techniques such as microarrays or qRT-PCR to date captures the most abundant conserved transcripts but precludes global analysis of less abundant miRNAs.

miRNAs are short non-coding RNA sequences that bind to complementary mRNAs to regulate transcript stability, usually by destabilizing mRNA poly-A tails which leads to transcript degradation (Bartel, 2018). Some miRNAs have cell type specificity during development and contribute to post-transcriptional regulation to maintain the equilibrium between mRNA transcription and degradation (Nowakowski et al., 2018). Sorting of miRNA cargo into EVs is controlled in part by target mRNA abundance which determines the dispensability of certain miRNAs in a passive mechanism of miRNA disposal inside EVs (Squadrito et al., 2014). At the same time, EV cargo miRNAs contain specific RNA-binding protein (RBP) sequence motifs. RBPs bind these sites to guide miRNAs in the MVB/endosomal compartment to remain in the cell or be sorted via the EXOMotif or other sequences into EVs as cargo (Garcia-Martin et al., 2022).

Here, we generated astrocytes from human iPSC-derived NPCs using a fast differentiation protocol yielding functional astrocytes that secrete ADEVs which can be isolated using ultracentrifugation. Analysis of miRNA cargo using RNA-seq showed that ADEVs contain significantly enriched miRNAs that participate in pathways regulating neuronal activity. XStreme motif discovery analysis unveiled the presence of RBP sequence motifs that are enriched on ADEV-miRNAs, supporting a sequence-dependent miRNA loading mechanism in ADEVs. Indeed, the most differentially sorted miR-483-5p cargo contains a previously described EXOmotif and is known to target MECP2, HDAC4 and TBLX1 whose protein products interact as cofactors (Han et al., 2013). miR-483-5p is depleted in peripheral blood of young RTT patients and has been proposed to be a candidate biomarker for early diagnostic screening (Castells et al., 2021). We speculate that miR-483-5p may be a potential prognostic biomarker of astrocyte function.