Journal: Neural Regeneration Research

Article Title: Human neural stem cell–derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01144

Figure Lengend Snippet: Characterization of hNSCs and hNSC-EVs. (A) Morphology of first- and third-passage hNSCs. Scale bar: 100 μm. (B) The hNSCs were positive for neural stem cell markers. Representative images of hNSCs showing PAX6 in purple, Nestin in purple, and DAPI (nuclear counterstain) in blue. Scale bar: 50 μm. (C) Analysis of NSC multipotentiality via a standard differentiation assay showing upregulation of neuronal and neuroglial markers. Representative images of hNSCs showing βIII-tubulin (neuron) in white, GFAP (neuroglia) in green, and DAPI (nuclear counterstain) in blue. Scale bar: 50 μm. (D) Nanoparticle tracking analysis showing the diameters of the hNSC-EVs. (E) TEM showing hNSC-EV morphology. Scale bar: 100 nm. (F) Immunoblot for CD9, CD63, TSG101, and calnexin expression by hNSC-EVs. (G) hNSC-EV internalization by HUVECs, mouse hippocampal neuronal cells (HT22), and mouse microglial cells (BV2). Representative confocal microscope images of cells with green-labeled cytoplasmic and DAPI-stained nuclei (blue) 6 hours after exposure to PKH26-labeled hNSC-EVs (red). Scale bar: 5 μm. All experiments were performed at least three times. DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; hNSC: human neural stem cell; hNSC-EVs: hNSC-derived extracellular vesicles; HUVEC: human umbilical vein endothelial cells; NTA: nanoparticle tracking analysis.

Article Snippet: HT22 cell viability was measured using a Cell Counting Kit-8 (CCK8) kit (CK04, Dojindo, Tabaru, Japan) according to the manufacturer’s instructions.

Techniques: Differentiation Assay, Western Blot, Expressing, Microscopy, Labeling, Staining, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Human neural stem cell–derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01144

Figure Lengend Snippet: Validation of hNSC-EV efficacy in vitro. (A) Morphology of HT22 cells. Scale bars: 100 μm. (B) Decreased toxic damage and (C) increased cell survival were observed in the hNSC-EV group, as assessed by lactate dehydrogenase assay, and Cell Counting Kit-8 assay, respectively. (D) Flow cytometry analysis of the effect of hNSC-EVs on HT22 cell apoptosis. (E) Percentage of annexin V–positive (apoptotic) cells. (F, G) Representative immunofluorescence images and quantification of ROS (green). Scale bars: 100 μm. The “Normal” group represents untreated HT22 cells, the “Control” group comprises HT22 cells exposed to 200 μM H 2 O 2 , and the “EVs” group consists of HT22 cells exposed to 200 μM H 2 O 2 , followed by treatment with hNSC-EVs. All experiments were performed at least three times. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparisons test. hNSC: Human neural stem cell; hNSC-EVs: hNSC-derived extracellular vesicles; ROS: reactive oxygen species.

Article Snippet: HT22 cell viability was measured using a Cell Counting Kit-8 (CCK8) kit (CK04, Dojindo, Tabaru, Japan) according to the manufacturer’s instructions.

Techniques: In Vitro, Lactate Dehydrogenase Assay, Cell Counting, Flow Cytometry, Immunofluorescence, Control, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Human neural stem cell–derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

doi: 10.4103/NRR.NRR-D-23-01144

Figure Lengend Snippet: Effect of hNSCs-EVs on PI3K/AKT/mTOR signaling pathway components in the context of oxidative stress. (A) Heatmap showing differentially expressed genes in HT22 cells. HT22 were incubated with H 2 O 2 , followed by hNSCs-EV treatment. (B) Gene set enrichment analysis (GSEA) was performed to compare the expression of genes involved in response to reactive oxygen species, the G2M checkpoint, wound healing, and PI3K/AKT/mTOR signaling between the hNSCs-EV group and the H 2 O 2 group. (C) p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, caspase 3, and cleaved-caspase 3 expression in HT22 cells, GAPDH was chosen as an internal reference for protein normalization. (D) Statistical analysis showing differences in p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and cleaved caspase 3 expression levels among the groups. The “Normal” group represents untreated HT22 cells, the “Control” group comprises HT22 cells exposed to 200 μM H 2 O 2 , and the “EVs” group consists of HT22 cells exposed to 200 μM H 2 O 2 , followed by treatment with hNSC-EVs. The “EVs + Inhibitor” group consisted of neural cells exposed to 200 μM H 2 O 2 and 100 nM dactolisib (PI3K inhibitor), followed by treatment with hNSC-EVs. All experiments were performed at least three times. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparisons test. AKT: Protein kinase B; hNSC: human neural stem cell; hNSC-EVs: hNSC-derived extracellular vesicles; mTOR: mammalian target of rapamycin; p-: phosphorylated-; PI3K: phosphoinositide 3-kinase.

Article Snippet: HT22 cell viability was measured using a Cell Counting Kit-8 (CCK8) kit (CK04, Dojindo, Tabaru, Japan) according to the manufacturer’s instructions.

Techniques: Incubation, Expressing, Control, Derivative Assay