Journal: PLoS ONE

Article Title: Engrafted Human Induced Pluripotent Stem Cell-Derived Anterior Specified Neural Progenitors Protect the Rat Crushed Optic Nerve

doi: 10.1371/journal.pone.0071855

Figure Lengend Snippet: Retrograde labeling by intracollicular injection of DiI showed labeled retinal ganglion cells (RGCs) in the retina. (A) Representative micrographs of retrograde-labeled RGCs in different experimental groups including (A I ) animals with intact optic nerve; (A II ) animals with optic nerve crush (ONC); (A III ) animals with ONC and transplanted cells; and (A IV ) animals with ONC and treated with CM. For higher magnification images, please see . (B) Quantitative analysis of RGC survival as number of cells/mm 2 in the retina of different groups on day 21 post-ONC, n = 4, a: p< 0.05 as determined by student t -test. Compared to the intact nerve, a few cells were labeled in the eye with vehicle-transplanted nerve following the crush. Both hiPSC-NPs transplantation and CM of cells (CM-hiPSC-NPs) protected RGCs and increased the remaining cell population. (C) CM of hiPSC-NPs evaluated by ELISA for the secretion of trophic factors. CNTF, FGF2 and IGF1 were released into the medium. Data are expressed as mean±SEM, n = 9 independent experiments.

Article Snippet: To evaluate growth factor secretion by hiPSC-NPs, we quantified the CM of NPs for protein levels of ciliary neurotrophic factor (CNTF), bFGF and insulin-like growth factor 1 (IGF1) using specific ELISA kits (R&D Systems; DNT00 for CNTF, DFB50 for bFGF, DG100 for IGF1) according to the manufacturer’s instructions.

Techniques: Labeling, Injection, Transplantation Assay, Enzyme-linked Immunosorbent Assay

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 1 Retinal wholemounts and sections from non-injured rats that received intravitreal AAV vector injections. (a and b) AAV-GFP injected rats. These rats also received multiple fluorogold (FG) injections into contralateral superior colliculus 5 weeks after the AAV-GFP eye injection. (a) Close correspondence between bIII-tubulin immunostaining (red) and retrograde FG label (gold) in RGCs. (b) Retinal wholemount immunostained for both GFP (green) and bIII-tubulin (red). Examples of retinotectally projecting ganglion cells triple labeled with FG, GFP and bIII-tubulin are shown by the arrowheads; occasional GFP+ cells in the ganglion cell layer were not labelled with FG or bIII- tubulin (arrows) and were presumably displaced amacrine cells. Note in this particular retinal region not all RGCs were FG+. (c–e) Transduced RGCs in retinas 11 weeks after intravitreal AAV-CNTF-GFP injection. (c) Wholemount stained with GFP (green) and bIII-tubulin (red). (d) Section immunostained with CNTF antibody (immunoreactive RGCs are arrowed). (e) Section immunostained for both CNTF (red) and GFP (green). Examples of (f) BDNF and (g) GAP-43 immunoreactivity in RGCs in retinas from AAV-BDNF-GFP and AAV-GAP43-GFP injected eyes respectively. Scale bars: a and c ¼ 25 mm (bar shown on a) b ¼ 25 mm; e–g ¼ 20 mm (bar shown on g).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Plasmid Preparation, Injection, Eye Injection, Immunostaining, Labeling, Staining

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 2 Retinal tissue from (a and e) AAV-CNTF-GFP, (b, c and f) AAV-BDNF-GFP, (d) AAV-GFP, or (g) AAV-GAP43-GFP treated animals, 7 weeks following optic nerve crush. To identify surviving and transduced RGCs, retinal wholemounts (a and b) and sections (c) were immunostained for both bIII-tubulin (red) and GFP (green). (d–g) fields from representative bIII-tubulin stained wholemounts after different AAV vector injections. These fields were taken from similar retinal eccentricities, about 1–1.5 mm from the optic disk. For each AAV vector, the mean numbers (7s.e.m.) of all surviving (bIII-tubulin+) RGCs and viable bIII-tubulin+ RGCs that were also GFP+ are shown in h. Abbreviations: , ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL). Scale bars: a–c ¼ 50 mm (bar shown on c); d–g ¼ 100 mm (bar shown on g).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Staining, Plasmid Preparation

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 3 Longitudinal cryosections of optic nerves 7 weeks after optic nerve crush and 8 weeks after intravitreal injection of AAV-GFP (a–c), AAV-BDNF-GFP (d–f) or AAV-CNTF-GFP (g–i). Fields in column one (a, d, g) are shown at higher magnification in column two (b, e, h). Columns two and three are the same fields viewed through different filter blocks. Each section was immunoreacted with bIII-tubulin and GFP antibodies to identify RGC axons proximal to the crush site. Arrowheads in a, d and g show the location of the crush. The bIII-tubulin+ axons arrowed in b, e, h are also visible in c, f, and i, indicating that these particular GFP+ axons originated from AAV-transduced RGCs. Scale bars: a, d, g ¼ 100 mm; b, c, e, f, h, i ¼ 50 mm (bar shown on b and c).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Injection

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 4 Longitudinal optic nerve sections from an AAV-CNTF-GFP injected rat, immunostained for bIII-tubulin (a–c) or GAP43 (d–e). (a) Many bIII-tubulin+ RGC axons traverse the crush site (arrowheads) and regenerate through the white matter of the distal optic nerve. (b) An adjacent section to A showing regrown bIII-tubulin+ axons at higher magnification. (c) Small fascicle of bIII-tubulin+ axons (arrowhead) with abnormal axonal branching (arrows) towards the middle of the optic nerve, 6.5 mm distal to the crush. (d) GAP43+ axons distal to the crush site. (e) GAP43+ axons at the optic chiasm. Outlined area in e is shown under higher magnification as an insert within e; a few regenerate RGC axons (arrows) are visible. LON, left (crushed) optic nerve; RON, right (uninjured) optic nerve. Scale bars: a ¼ 200 mm; b, d ¼ 100 mm (bar shown on b); c ¼ 50 mm; e ¼ 100 mm.

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Injection

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 5 (a and b) Retinal wholemounts from PN grafted rats, immunostained for bIII-tubulin. There were fewer surviving RGCs in AAV- GFP (a) Compared to AAV-CNTF-GFP (b) injected eyes. (c) Retina from AAV-CNTF-GFP eye. RGCs with regenerating axons were retrogradely labeled after fluorogold (FG) injection into the PN graft. Note the variation in soma size of FG+ RGCs. (d) GFP and FG label in retinal wholemount from another PN grafted animal. The three AAV-CNTF-GFP transduced RGCs (arrows) are not retrogradely labeled with FG. (e) Longitudinal section of PN graft from a rat that received an intravitreal injection of AAV-CNTF-GFP. Many bIII-tubulin+ axons can be seen (Cy3 – red), a few of which are co-stained for GFP (FITC, axons appear yellow). Scale bars: a, b ¼ 100 mm; c, e ¼ 50 mm; d ¼ 25 mm.

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Injection, Labeling, Staining

Journal: Gene therapy

Article Title: AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

doi: 10.1038/sj.gt.3302791

Figure Lengend Snippet: Figure 7 Linear maps of the AAV vector plasmids (based on pTRUF12.1) used in these studies. With the exception of the inverted terminal repeats (ITR) all viral genes have been removed and replaced with (a) GFP, or (b) CNTF (with NGF secretory sequence, NGFss-CNTF), BDNF or GAP43 (all represented by X) and GFP, all under the control of a cyglomegalovirus/chicken b-actin hybrid promoter. Abbreviation: CMV (cyglomegalovirus promoter); IRES (internal ribosomal entry site) from poliovirus; SV40polyA (SV40 virus polyadenylation signal).

Article Snippet: Series of retinal sections were immunostained using monoclonal antibodies to bIIItubulin (1:1000, TUJ-1 Covance), rat GAP43 (1:2000, Chemicon) and/or polyclonal antibodies to GFP (1:300, Chemicon), CNTF (recognizes mouse and rat, 1:100, R&D Systems, MN, USA) or BDNF (recognizes rat and human, 1:300, Chemicon).

Techniques: Plasmid Preparation, Sequencing, Control, Virus

Journal: International Journal of Molecular Sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling

doi: 10.3390/ijms23158335

Figure Lengend Snippet: The tripartite CNTF-receptor complex is up-regulated by ciliary neurotrophic factor in cementoblasts. ( A , B ) WB showed protein expression of the CNTF-receptors (CNTFRα, LIFR and IL-6Rα) in OCCM-30 cells induced by CNTF protein (400 ng/mL) for various periods. Internal β-actin serves as loading control. ( C ) The expression of mRNAs encoding the CNTF-receptors were quantified by RT-qPCR. The relative mRNA expression of each gene was obtained through normalizing to internal PPIB . The statistical significance was determined by student t -test ( n = 3 for each group). ( D , E ) The CNTF-receptors immunofluorescent localization showed the expression of CNTFRα (red arrow), LIFR (yellow arrow) and IL-6Rα (orange arrow) in CNTF-treated OCCM-30 cells. Nuclei are stained with DAPI (blue). Scale bar: 100 μm (image magnification: 40×); 50 μm (image magnification: 60×). Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (* p < 0.05; ** p < 0.005).

Article Snippet: OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals).

Techniques: Expressing, Control, Quantitative RT-PCR, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling

doi: 10.3390/ijms23158335

Figure Lengend Snippet: Ciliary neurotrophic factor triggers GP130 protein expression and phosphorylated GP130 in cementoblasts. ( A , B ) The protein expression of GP130 and phosphorylated GP130 were determined by WB. Internal β-actin serve as loading control. The line chart shows the densitometric analysis of p-GP130 expression related to total GP-130 expression. ( C ) RT-qPCR quantification of GP130 ( IL-6st ) gene expression in OCCM-30 cells when treated with CNTF (400 ng/mL) for indicated time. The relative mRNA expression was obtained through normalizing to internal PPIB . ( D , E ) IF staining of subcellular localization of GP130 (white arrow) as well as p-GP130 (grey arrow) in non-stimulated cells (negative control) and CNTF-stimulated OCCM-30 cells. Nuclei are stained with DAPI (blue). Individual and merged images of GP130 and p-GP130 are shown. Scale bar: 100 μm (image magnification: 40×); 50 μm (image magnification: 60×). Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (* p < 0.05; ** p < 0.005; *** p < 0.0005).

Article Snippet: OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals).

Techniques: Expressing, Control, Quantitative RT-PCR, Gene Expression, Staining, Negative Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling

doi: 10.3390/ijms23158335

Figure Lengend Snippet: Ciliary neurotrophic factor impairs OCCM-30 homeostasis and activates the expression of ERK1/2 MAPK signaling. ( A , B ) Immunofluorescence microscopy images show representative proliferation markers Ki-67 expression. OCCM-30 cells were exposed to CNTF (400 ng/mL) and then underwent immunofluorescence staining for Ki-67 to visualize cells in the proliferation stage. The proportion of proliferating cells for each group was quantified according to Ki-67 positive cells (Ki-67 + )/total cell counting (DAPI). ( C ) Cell viability assay was performed by MTS assay. IL-6 cytokine served as positive control. ( D , E ) Representative immunoblot of p-ERK1/2 protein expression in the presence of CNTF (400 ng/mL) at different time points. Internal β-actin serves as loading control. Densitometric immunoblot analysis of bands indicated the enhanced p-ERK1/2 expression relative to that of the control group. Densitometric results are showed as fold change. Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (* p < 0.05; ** p < 0.005; *** p < 0.0005).

Article Snippet: OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Cell Counting, Viability Assay, MTS Assay, Positive Control, Western Blot, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling

doi: 10.3390/ijms23158335

Figure Lengend Snippet: ERK1/2 signal is involved in the regulation of apoptosis of cementoblasts and the caspases pathway. ( A , B ) Graphics show the percentages of apoptotic cells exposed to ERK1/2 inhibitor (1.0 μg/mL, FR180204) as well as co-stimulation with CNTF (400 ng/mL). ( C ) The scheme summarizes the mode of CNTF action in cementoblasts: CNTF activated the tripartite CNTF-receptor complex targets and phosphorylated GP130 protein, which recruits ERK1/2 signaling and caspases signaling expression. FR180204 promotes apoptosis in OCCM-30 cells and CNTF addition suppressed the ERK1/2 inhibitor-induced apoptosis within a short-term period. Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (** p < 0.005; *** p < 0.0005).

Article Snippet: OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals).

Techniques: Expressing, Standard Deviation