Journal: Scientific Reports

Article Title: Immune-tolerance to human iPS-derived neural progenitors xenografted into the immature cerebellum is overridden by species-specific differences in differentiation timing

doi: 10.1038/s41598-020-79502-9

Figure Lengend Snippet: ( a ) Western blot analysis. After 55 days of in vitro differentiation treatment of eGFP expressing hiPSdNP the levels of antigens related to cellular proliferation and neural and/or glial differentiation change. The proliferation related Ki67 antigen decreases sharply in DhiPSdNP compared to hiPSdNP. Similarly, in DhiPSdNP Nestin and SOX2 both labeling neuro-glial precursors decrease, while MAP2 and β-III-tubulin that are present in postmitotic neurons, and GFAP, that is mainly present in astrocytes, increase. A housekeeping protein like β-Actin, remains the same. Numbers indicate the apparent molecular weight in kilodaltons. An uncropped image of the westerns used to assemble Fig. 3a is provided in Fig. . ( b ) Splenocytes from CD1 mice immunized with hiPSdNP or DhiPSdNP, or not immunized (Naive), were stimulated with PMA/ionomycin, in the presence of brefeldin A, and tested for IFNγ production by intracellular staining and flow cytometry. The histograms report the quantification of IFNγ production by CD4 + T lymphocytes; bars: average. Values are subtracted of background, i.e. spontaneous IFNγ release by unstimulated CD4 + or CD8 + T cells. N = 3 mice per group. One-way ANOVA followed by Tukey’s test: * p < 0.05. ( c ) Same as in B but the histogram shows the quantification of IFNγ production by CD8 + T lymphocytes. ( d ) hiPSdNP and DhiPSdNP were tested in vitro for suppressive activity against T cells. Responder CFSE-labeled splenocytes were stimulated with anti CD3 and anti CD28 antibodies, and left alone (+) or in the presence of hiPSdNP/DhiPSdNP, and tested after 5 days by flow cytometry. hiPSdNP/DhiPSdNP: responder ratio 1:1, 1:1.5 or 1:2 as indicated. Histogram reports percentage of proliferation for CD4 + T lymphocytes. Negative (−) controls were unstimulated splenocytes. The experiment was repeated three times. One-way ANOVA followed by Tukey’s test: ** p < 0.005. ( e ) Same as in D but we measured proliferation of CD8 + T lymphocytes. ( f ) Kaplan–Meier analysis of survival of hiPSdNP transplanted in immunocompetent CD1 mice (CD1) and Wistar rats (WR) or immunodeficient mice (NOD-SCID) alone or as a mixture with unlabeled DhiPSdNP (hiPSdNP + N) or with adult rat cerebellar cells (hiPSdNP + Cb). ( g ) Mouse colliculum P45. Mice were transplanted at P3 with a mixture of EGFP labelled hiPSdNP and unlabelled DhiPSdNP. Dapi staining shows that the EGFP positive cells are mono-nucleated and their chromatin condensation pattern is very different (arrow) from that of the host cells (arrows). Scale bar: 10 µm. ( h ) Mouse cerebellum and brainstem 90 after transplant. The animal was transplanted in utero with a mixture of eGFP positive hiPSdNP and a suspension of cells derived by mechanical dissociation of adult rat cerebellum. Sagittal section eGFP and Dapi counterstained. The main cluster of transplanted cells is localized in the superior colliculus (SC) but eGFP positive cells are present also in the inferior colliculus (IC) and the brainstem. GL: granular layer, ML: molecular layer. Scale bar: 100 µm. On the right an enlargement of the area of the transplant. eGFP positive cells show different degrees of morphological differentiation and some display glial and neuronal morphologies. Scale bar: 25 µm. ( i ) Same as in h. Arrows indicate eGFP cells showing hNCAM immunopositivity. Scale bar: 15 µm.

Article Snippet: The following primary antibodies that were not mentioned before were used for western blotting included: mouse monoclonal anti-Ki-67 1:1000 (Agilent Dako #M7240); mouse monoclonal anti-MAP2 Merck 1:5000 (Millipore MAB3418); mouse monoclonal anti-nestin 1:1000 (Merck Millipore MAB5326); mouse anti-βIII-Tubulin (TUJ1) 1:10,000 (Sigma-Aldrich T8578); mouse anti-GFAP 1:2000 (Merck Millipore AB5804); mouse monoclonal anti-β-Actin 1:20,000 (Sigma-Aldrich A1978); mouse monoclonal anti-SOX2 1:1000 (Cell Signaling Technology 3579).

Techniques: Western Blot, In Vitro, Expressing, Labeling, Molecular Weight, Staining, Flow Cytometry, Activity Assay, In Utero, Derivative Assay

Journal:

Article Title: In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

doi: 10.1128/JVI.77.18.9799-9808.2003

Figure Lengend Snippet: Gene transduction into primary rat cerebellar cells with AcVSVG-CAGFP. Primary rat cerebellar and hippocampal cultures were infected with AcVSVG-CAGFP. Immunofluorescence was examined by confocal microscopy. (A, D, and G) Anti-GFP immunochemistry. (B) Anti-Calbindin immunochemistry as a purkinje marker. (E) Anti-GFAP immunochemistry as a glial marker. (H) Anti-MAP2 immunochemistry as a neuronal marker. C, F, and I are merged images.

Article Snippet: Sections were further examined by immunohistochemical analysis after staining with either rabbit anti-GFP (Molecular Probes), mouse anti-MAP2 (ICN Biomedicals), or mouse anti-GFAP (Zymed Laboratories) as described above.

Techniques: Transduction, Infection, Immunofluorescence, Confocal Microscopy, Marker

Journal:

Article Title: In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

doi: 10.1128/JVI.77.18.9799-9808.2003

Figure Lengend Snippet: GFP expression in mouse brains after cerebral injections of AcVSVG-CAGFP. Mice were injected with 4 × 107 PFU of AcVSVG-CAGFP in the right lateral ventricle. GFP expression in the brain was examined by fluorescent stereomicroscopy 2 days after injection. (A) Panels A to D are stereomicroscopic images of whole brain (A and B) and brain cross sections (C and D). Panels A and C are bright-field views, while panels B and D are fluorescent views. Arrows and dark staining indicate the injection route, as the infiltrated viral inoculum contained 0.04% trypan blue. (B) Immunohistochemical staining of the cryostat sections was examined by fluorescence microscopy following staining with antibodies specific for GFP (A and D), GFAP as a glial marker (B), or MAP2 as a neuronal marker (E). Panels C and F are merged images.

Article Snippet: Sections were further examined by immunohistochemical analysis after staining with either rabbit anti-GFP (Molecular Probes), mouse anti-MAP2 (ICN Biomedicals), or mouse anti-GFAP (Zymed Laboratories) as described above.

Techniques: Expressing, Injection, Staining, Immunohistochemical staining, Fluorescence, Microscopy, Marker