Journal: Scientific Reports

Article Title: Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

doi: 10.1038/s41598-017-13764-8

Figure Lengend Snippet: TSP1 stimulated expression of the synaptic protein SV2 along connections between EGFP-ScNs and CN neurons. ( a ) Immunofluorescence showed that TUJ1-expressing connections were found between EGFP-ScNs (asterisk) and wild type CN neurons (arrowhead). The number of SV2 puncta (arrows) along connections increased when 2–20 nM TSP1 was supplied. ( b ) The quantitative study suggests that the 10 nM TSP1 group exerted significant effects to the expression of SV2 puncta, including relative number and area of SV2 puncta (mean ± standard error shown in the figure; **indicates P < 0.01; ANOVA followed by Tukey post hoc test). Scale bar: 10 µm in ( a ).

Article Snippet: For immunodepletion, Dynabeads (Invitrogen) were incubated with anti-TSP1 antibody (25 μg/ml, R&D system) or mouse IgG whole protein (25 μg/ml, Jackson ImmunoResearch; the mock control) at room temperature for 30 min.

Techniques: Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

doi: 10.1038/s41598-017-13764-8

Figure Lengend Snippet: TSP1 immunodepletion or TSP1 knockout abolished ACM-exerted effects, which was rescued by the supplementation of exogenous TSP1 protein. ( a ) TSP1 antibodies were used for western blotting, which showed clear TSP1 bands in TSP1, mock, and ACM groups, a weak band in TSP1 immunodepleted ACM group (TSP1-ID-ACM), and no bands in empty, DMEM/F12, or TSP1 knockout ACM (TSP1-KO-ACM) groups. The image was cropped and inverted from a digital image captured by UVP imaging system (original images with ladder in the supplemental Fig. ). ( b ) Immunofluorescence shows TUJ1-expressing connections between EGFP-ScNs (asterisks) and wild type CN neurons (arrowheads). A number of SV2-expressing puncta were observed along connections in the mock group (co-cultures supplied with the wild type ACM that has been immunodepleted by mouse IgG). The number of SV2-expressing puncta (arrows) along connections was reduced in the TSP1-ID-ACM group (co-cultures supplied with TSP1-ID-ACM), whereas it was increased when exogenous TSP1 protein (10 nM) was supplemented (the rescue group). ( b1 ) The quantitative study indicates that the relative number and area of SV2 puncta decreased in the TSP1-ID-ACM group, whereas they significantly increased in the rescue group (mean ± standard error shown in the figure; **indicates P < 0.01; ANOVA followed by Tukey post hoc test). ( c ) Immunofluorescence showed TUJ1-expressing connections between EGFP-ScNs (asterisks) and wild type CN neurons (arrowheads). A few SV2-expressing puncta (arrows) were observed along connections in the control (co-cultures without ACM supplement) and TSP1-KO-ACM groups (ACM collected from TSP1 knockout mice). In contrast, many SV2-expressing puncta were found along connections in the rescue group (co-cultures supplies with TSP1-KO-ACM in the presence of exogenous 10 nM TSP1 protein).( c1 ) The quantitative study shows that relative number and area of SV2 puncta along connections were lower in the control and TSP1-KO-ACM groups, whereas they were significantly higher in the rescue group. Statistical analysis showed significant difference (mean ± standard error shown in the figure; *indicates P < 0.05; ANOVA followed by Tukey post hoc test). Scale bar: 20 µm in (b), and 10 µm in ( c ).

Article Snippet: For immunodepletion, Dynabeads (Invitrogen) were incubated with anti-TSP1 antibody (25 μg/ml, R&D system) or mouse IgG whole protein (25 μg/ml, Jackson ImmunoResearch; the mock control) at room temperature for 30 min.

Techniques: Immunodepletion, Knock-Out, Western Blot, Imaging, Immunofluorescence, Expressing, Control

Journal: Scientific Reports

Article Title: Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

doi: 10.1038/s41598-017-13764-8

Figure Lengend Snippet: New connections between EGFP-ScNs and CN neurons expressed synaptic proteins. ( a ) TUJ1-positive connections were observed between EGFP-ScNs and wild type CN neurons, which also expressed presynaptic marker synapsin and postsynaptic marker PSD93 in the control (Ctrl; co-culture without supplement), TSP1, and ACM groups. Confocal microscopy-based co-localization showed obvious apposition of synapsin and PSD93 in the control, ACM and TSP1 groups (Person’s correlation 0.718 ± 0.057, Pearson’s R test, Leica LAS AF Lite Co-localization software). ( b ) The quantitative study showed that relative number and area of puncta along connections were lower in the control group, whereas they were significantly higher in the ACM and TSP1 groups (mean ± standard error shown in the figure; *indicates P < 0.05, **indicates P < 0.01, ANOVA followed by Tukey post hoc test). Scale bar: 10 µm in ( a ).

Article Snippet: For immunodepletion, Dynabeads (Invitrogen) were incubated with anti-TSP1 antibody (25 μg/ml, R&D system) or mouse IgG whole protein (25 μg/ml, Jackson ImmunoResearch; the mock control) at room temperature for 30 min.

Techniques: Marker, Control, Co-Culture Assay, Confocal Microscopy, Software

Journal: Scientific Reports

Article Title: Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

doi: 10.1038/s41598-017-13764-8

Figure Lengend Snippet: New connections between EGFP-ScNs and CN neurons expressed glutamatergic proteins. ( a ) TUJ1-expressing connections were seen between EGFP-ScNs (asterisks) and wild type CN neurons (arrowheads), which also expressed the glutamatergic marker VGluT1 in the control (Ctrl), TSP1, and ACM groups. ( b ) The quantitative study revealed that relative number and area of VGluT1 puncta along connections were lower in the control group, whereas they were significantly higher in the ACM and TSP1 groups (mean ± standard error shown in the figure; *indicates P < 0.05, **indicates P < 0.01, ANOVA followed by Tukey post hoc test). Scale bar: 10 µm in ( a ).

Article Snippet: For immunodepletion, Dynabeads (Invitrogen) were incubated with anti-TSP1 antibody (25 μg/ml, R&D system) or mouse IgG whole protein (25 μg/ml, Jackson ImmunoResearch; the mock control) at room temperature for 30 min.

Techniques: Expressing, Marker, Control

Journal: Scientific Reports

Article Title: Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons

doi: 10.1038/s41598-017-13764-8

Figure Lengend Snippet: The synaptic vesicle recycling study of connections between EGFP-ScNs and CN neurons. ( a ) EGFP-ScNs and CN neurons were co-cultured for 4–6 days, followed by FM4-64 synaptic vesicle recycling study. In initial exposure to FM4-64 for 1 min, no obvious staining was found in 3 groups. After high potassium stimulation, obvious staining was found in the soma and neurite outgrowths in the ACM and TSP1 groups. In the recovery stage, significant vesicle staining was observed in soma and nerve terminals of ACM and TSP1 groups. During rinse stages, the staining of vesicles remained distinct in the soma and neurite outgrowths. ( b ) The puncta at the recovery stage was used for the quantitative study. It was observed that relative number and area of FM4-64 vesicle along connections were lower in the control group, whereas they were significantly higher in ACM and TSP1 groups (mean ± standard error shown in the figure; **indicates P < 0.01, ANOVA followed by Tukey post hoc test). Scale bar: 25 µm in ( a ).

Article Snippet: For immunodepletion, Dynabeads (Invitrogen) were incubated with anti-TSP1 antibody (25 μg/ml, R&D system) or mouse IgG whole protein (25 μg/ml, Jackson ImmunoResearch; the mock control) at room temperature for 30 min.

Techniques: Cell Culture, Staining, Control