Journal: Neural Regeneration Research

Article Title: Human-induced pluripotent stem cell–derived neural stem cell exosomes improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

doi: 10.4103/NRR.NRR-D-23-01889

Figure Lengend Snippet: hiPSC–NSC–Exos modulate astrocyte activation and the inflammatory response post-ICH. (A, B) Representative immunofluorescence staining images and quantification of A1 astrocyte markers C3 (red, AlexaFluor 594) and GFAP (green, AlexaFluor 488) in brain tissue near the lesion ( n = 6). The proportion of A1 astrocytes (C3 + GFAP + cells) in mice treated with hiPSC–NSC–Exos was significantly lower. (C, D) Representative immunofluorescence staining images and quantification of A2 astrocyte markers S100β (red, AlexaFluor 594) and GFAP (green, AlexaFluor 488) in brain tissue near the lesion ( n = 6). The proportion of A2 astrocytes (S100β + GFAP + cells) in mice treated with hiPSC–NSC–Exos was significantly higher. (E) Intracranial cytokine expression profiles of ICH mice treated with PBS or Exo, as assessed by Luminex liquid chip ( n = 3). (F, G) Representative immunofluorescence staining images and quantification of MCP-1 (red, AlexaFluor 594) and GFAP (green, AlexaFluor 488) in brain tissue near the lesion ( n = 6). Scale bars: 50 μm, 20 μm for low and high magnification, respectively. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test). All statistical values are presented in . DAPI: 4′,6-Diamidino-2-phenylindole; Exo: exosomes; GFAP: glial fibrillary acidic protein; hiPSC: human induced pluripotent stem cell; ICH: intracerebral hemorrhage; MCP-1: monocyte chemoattractant protein-1; NSC: neural stem cell; PBS: phosphate-buffered saline.

Article Snippet: After blocking with 3% bovine serum albumin for 1 hour, the brain cryosections were incubated overnight at 4°C in a humidified environment with the antibodies to the following proteins: CD31 (mouse, 1:300, Abcam, Cat# ab9498, RRID: AB_307284), zona occludens-1 (ZO-1, rabbit, 1:300, Cell Signaling Technology, Cat# 13663, RRID: AB_2798287), glial fibrillary acidic protein (GFAP, mouse, 1:500, Cell Signaling Technology, Cat# 3670, RRID: AB_561049), and monocyte chemoattractant protein-1 (MCP-1, mouse, 1:500, Novus Biologicals, Littleton, CO, USA, Cat# NBP2-22115, RRID: AB_2728754).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Luminex, Saline

Journal: Neural Regeneration Research

Article Title: Human-induced pluripotent stem cell–derived neural stem cell exosomes improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

doi: 10.4103/NRR.NRR-D-23-01889

Figure Lengend Snippet: hiPSC–NSC–Exos enhance neurological deficits and alleviate brain edema after ICH via activation of the PI3K-AKT signaling pathway. (A) Scatter plot of differentially expressed genes in the affected hemisphere after ICH ( n = 3). (B) Gene Ontology (GO) analysis of differentially expressed genes. (C) Kyoto Encyclopedia of Genes and Genomic (KEGG) pathway enrichment analysis of the top 10 differentially expressed genes, with a focus on the PI3K-AKT signaling pathway. (D–F) Representative western blotting and quantitative analysis of p-PI3K, PI3K, p-AKT, and AKT expression levels ( n = 6). (G–I) Neurobehavioral function tests (rotarod test, corner turn test, and mNSS) were conducted on days 1 and 3 after ICH ( n = 6). The longer fall latency in rotarod tests and the fewer ipsilateral turnings in the corner turn test indicated better neurological function. (J) Quantification of water content in the ipsilateral hemisphere, contralateral hemisphere, and cerebellum ( n = 6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. PBS group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hiPSC-NSC-Exo group (Student’s t -test [E, F] or repeated-measures two-way analysis of variance followed by least significant difference post hoc tests [G–J]). All statistical values are presented in . AKT: Protein kinase B; DMSO: dimethyl sulfoxide; Exo: exosomes; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hiPSC: human induced pluripotent stem cell; ICH: intracerebral hemorrhage; LY294002: a PI3K/AKT inhibitor; MCP-1: monocyte chemoattractant protein-1; mNSS: modified Neurological Severity Score; NSC: neural stem cell; p-AKT: phosphorylated protein kinase B; p-PI3K: phosphorylated phosphoinositide 3-kinase; PI3K: phosphoinositide 3-kinase; PBS: phosphate-buffered saline.

Article Snippet: After blocking with 3% bovine serum albumin for 1 hour, the brain cryosections were incubated overnight at 4°C in a humidified environment with the antibodies to the following proteins: CD31 (mouse, 1:300, Abcam, Cat# ab9498, RRID: AB_307284), zona occludens-1 (ZO-1, rabbit, 1:300, Cell Signaling Technology, Cat# 13663, RRID: AB_2798287), glial fibrillary acidic protein (GFAP, mouse, 1:500, Cell Signaling Technology, Cat# 3670, RRID: AB_561049), and monocyte chemoattractant protein-1 (MCP-1, mouse, 1:500, Novus Biologicals, Littleton, CO, USA, Cat# NBP2-22115, RRID: AB_2728754).

Techniques: Activation Assay, Western Blot, Expressing, Modification, Saline

Journal: Neural Regeneration Research

Article Title: Human-induced pluripotent stem cell–derived neural stem cell exosomes improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

doi: 10.4103/NRR.NRR-D-23-01889

Figure Lengend Snippet: hiPSC–NSC–Exos protect BBB integrity by regulating astrocytes rather than directly affecting endothelial cells. (A) Treatment of endothelial cells with hemin/exosomes. Created with Figdraw. (B) Endothelial cell survival rate under different hemin concentrations ( n = 6). (C, D) Endothelial cell leakage and TEER after astrocytes were exposed to exosomes at various concentrations ( n = 3). (E) Exosome treatment in a hemin-treated in vitro BBB model. Created with Figdraw. (F) Representative images of Transwell assay wells in the BBB model: F1-cell–free, F2-only lower astrocytes, which are smaller in size and exhibit a stellate or multipolar morphology, F3-only upper endothelial cells, which are larger and have a spindle-shaped or flattened polygonal appearance, and F4–co-culture of upper chamber endothelial cells, characterized by their larger, spindle-shaped or flat polygonal structures, and lower chamber astrocytes that are smaller and exhibit stellate or multipolar configurations. Scale bar: 200 μm. (G) Astrocyte survival rate under varying hemin concentrations ( n = 6). (H) MCP-1 expression level in BBB model medium ( n = 6). (I, J) EB leakage and TEER in the BBB model with different concentrations of exosomes ( n = 6). Data are presented as mean ± SEM. ** P < 0.01, *** P < 0.001, vs . PBS group (repeated-measures two-way analysis of variance followed by least significant difference post hoc tests). All statistical values are presented in . BBB: Blood-brain barrier; EB: Evans Blue; Exo: exosomes; hiPSC: human induced pluripotent stem cell; MCP-1: monocyte chemoattractant protein-1; NSC: neural stem cell; PBS: phosphate-buffered saline; TEER: transepithelial electrical resistance

Article Snippet: After blocking with 3% bovine serum albumin for 1 hour, the brain cryosections were incubated overnight at 4°C in a humidified environment with the antibodies to the following proteins: CD31 (mouse, 1:300, Abcam, Cat# ab9498, RRID: AB_307284), zona occludens-1 (ZO-1, rabbit, 1:300, Cell Signaling Technology, Cat# 13663, RRID: AB_2798287), glial fibrillary acidic protein (GFAP, mouse, 1:500, Cell Signaling Technology, Cat# 3670, RRID: AB_561049), and monocyte chemoattractant protein-1 (MCP-1, mouse, 1:500, Novus Biologicals, Littleton, CO, USA, Cat# NBP2-22115, RRID: AB_2728754).

Techniques: In Vitro, Transwell Assay, Co-Culture Assay, Expressing, Saline

Journal: Neural Regeneration Research

Article Title: Human-induced pluripotent stem cell–derived neural stem cell exosomes improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

doi: 10.4103/NRR.NRR-D-23-01889

Figure Lengend Snippet: hiPSC–NSC–Exos improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via the PI3K/AKT/MCP-1 axis. (A) Hemin/exosome + LY294002/DMSO treatment of astrocytes. (B–D) Representative western blot image and quantitative analysis of p-PI3K, PI3K, p-AKT, and AKT expression levels ( n = 6). (E) MCP-1 content in the medium ( n = 3). (F) LY294002 and C1142 treatment of the hemin-treated BBB model. (G) MCP-1 content in the medium of the BBB model ( n = 6). (H, I) EB leakage and TEER in the BBB model under different conditions ( n = 6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, vs . astrocyte group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . astrocyte + hemin group; && P < 0.01, &&& P < 0.001, vs . astrocyte + hemin + Exo group. All statistical values are presented in . AKT: Protein kinase B; BBB: blood–brain barrier; C1142: a MCP-1 neutralizing agent; DMSO: dimethyl sulfoxide; Exo: exosomes; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hiPSC: human induced pluripotent stem cell; LY294002: a PI3K/AKT inhibitor; MCP-1: monocyte chemoattractant protein-1; NSC: neural stem cell; p-AKT: phosphorylated protein kinase B; p-PI3K: phosphorylated phosphoinositide 3-kinase; PI3K: phosphoinositide 3-kinase.

Article Snippet: After blocking with 3% bovine serum albumin for 1 hour, the brain cryosections were incubated overnight at 4°C in a humidified environment with the antibodies to the following proteins: CD31 (mouse, 1:300, Abcam, Cat# ab9498, RRID: AB_307284), zona occludens-1 (ZO-1, rabbit, 1:300, Cell Signaling Technology, Cat# 13663, RRID: AB_2798287), glial fibrillary acidic protein (GFAP, mouse, 1:500, Cell Signaling Technology, Cat# 3670, RRID: AB_561049), and monocyte chemoattractant protein-1 (MCP-1, mouse, 1:500, Novus Biologicals, Littleton, CO, USA, Cat# NBP2-22115, RRID: AB_2728754).

Techniques: Western Blot, Expressing

Journal: Frontiers in Neurology

Article Title: Immuno-surveillance and protection of the human cochlea

doi: 10.3389/fneur.2024.1355785

Figure Lengend Snippet: Antibodies used for studies of the human cochlea and endolymphatic sac.

Article Snippet: CCL2 , Polyclonal , 1:500 , Rabbit , NBP1-07035 , Novus Biologicals.

Techniques: