Induced neural progenitor cell‐derived extracellular vesicles promote neural progenitor cell survival via extracellular signal‐regulated kinase pathway

Proposed model for the anti‐apoptotic effects of induced neural stem/progenitor cell (iNPC)‐derived extracellular vesicles (EVs). iNPC release EVs that are abundantly loaded with growth factor‐related proteins. These growth factor‐enriched EVs enhance the phosphorylation of extracellular signal‐regulated kinase (ERK), but not AKt. The EV‐induced activation of ERK pathway then inhibit the apoptosis of NPCs in various pathological conditions including oxidative stress and starvation.


Induced neural progenitor cell-derived extracellular vesicles promote neural progenitor cell survival via extracellular signalregulated kinase pathway
Extracellular vesicles (EVs) are phospholipid-bilayer-enclosed extracellular spherical structures that regulate a variety of cellular processes by horizontally transferring bioactive cargos. 1 Emerging evidence has implied neural stem/progenitor cell (NPC)-derived EVs (WT-EVs) as promising therapeutic strategy to replace stem cell transplantation in treating neurological disorders, especially for neurodegenerative diseases like Alzheimer's disease (AD) that are in lack of effective therapeutic drugs. 2 Unfortunately, the application of NPCs in mass production of EVs are restricted due to ethical/religious concerns, problematic logistics of acquiring fetal tissues, and potential autogenous immune response. 3 Hence, we reprogrammed somatic cells into induced NPCs (iNPCs) to avoid aforementioned issues. 4 Interestingly, EVs released from both fibroblast-derived iNPCs (F-EVs) and astrocyte-derived ones (A-EVs) exhibit higher potential in promoting NPC proliferation in vitro versus WT-EVs. 5 However, the roles of those EVs on neuroprotection remain unknown. We, therefore, examined the effects of NPC-and iNPC-derived EVs on NPC survival in vitro. Data are presented as the means ±SEM.
Shapiro-Wilk tests were used to evaluate the normality of the distribution. One-way ANOVA or Kruskal-Wallis ANOVA were used for data that are normally distributed or not normally distributed, respectively. Significance was set at p < 0.05.
To determine the roles of EVs on NPC survival, we first characterized EVs derived from NPCs and iNPCs. EVs were isolated from the conditioned medium (CM) of either NPCs or iNPCs using gradient centrifugation approach. Scanning electron microscopy indicated a great number of vesicles being released from the plasma membrane ( Figure S1A). Transmission electron microscopy revealed typical cup-shaped morphology of EVs with sizes less than 200 nm ( Figure   S1B). Western blotting identified EVs specific markers Flotillin-1 and HSP70 in EV lysates, confirming the purification of EVs ( Figure S1C).     control groups ( Figure 2E-H). Hence, our observations indicated that the anti-apoptotic effects of iNPC-derived EVs are mediated by ERK pathway.
The loss of brain cells, especially NPCs has been considered as a key pathological feature of neurological disorders. NPCs located in the adult subventricular and subgranular zone are attacked by stroke and directed to apoptosis before being induced to migrate to the lesion point and differentiate into neurons for replacing damaged cells. 6 The excessive expression of apoptotic genes including Bak have been found in various neurodegenerative diseases including AD, inflating apoptosis and causing mortality. 7,8 Our results demonstrated that iNPC-derived EVs significantly promoted NPC survival in various apoptosis models, suggesting EVs with a novel role in neuroprotection. However, as all experiments in the study were carried out in vitro, the therapeutic effects of iNPC-EVs on treating neurological disorders need to be verified in vivo. Inspiringly, promising results have been obtained by intravenously administrating iNPC-EVs into middle cerebral artery occlusion (MCAO) mouse models (data are not shown). In addition, ERK signaling, the key downstream regulator of iNPC-EVs, is one of the most essential intracellular pathways in cell death regulation. 9 The activation of ERK pathway significantly increases the viability of NPCs in stroke. 10 However, it is worth-noting that ERK pathway may exhibit dual roles in controlling the apoptosis of other types of brain cells like neurons. 9 Thus, the controversial roles of ERK pathway in apoptosis reveal that the anti-apoptotic effects of iNPC-derived EVs may not be applicable to all brain cells, which requires more investigations in the future. Besides, multiple research groups including us have reported the important roles of other EV contents, especially miRNAs, in mediating the physiological and pathological effects of EVs in the central nervous system or the peripheral nervous system (see reviews 1,[11][12][13] ). We have found that WT-EVs are enriched with miR-9 and miR-21. 14,15 These miRNAs in EVs significantly promote neurogenesis in vitro. 14,15 These findings implied the importance to examine the involvement of iNPC-EVcontaining miRNAs in the regulation of cell survival, which is under investigation. In summary, our study identified EVs as an essential element of iNPC-mediated modification of microenvironment and provided new evidence for the inextricable crosstalk between cell extrinsic and intrinsic factors in regulating NPC survival. Our findings not only unveiled a possible mechanism for the therapeutic effect of iNPC transplantation, but also implied iNPC-derived EVs as a promising cell-free therapy candidate to protect brain cells in neurological disorders.

CO N FLI C T O F I NTE R E S T S
The authors declare non-financial conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.