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Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival.

Ma J, Guo C, Guo C, Sun Y, Liao T, Beattie U, López FJ, Chen DF, Lashkari K - PLoS ONE (2015)

Bottom Line: Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects.In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss.RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, 02114, MA, United States of America.

ABSTRACT
We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.

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Quantification of RGC density using ß-III tubulin (red fluorescence) in retinal flatmounts under different experimental conditions.(A—E) Confocal images show significant and robust RGC loss is visualized in the saline, hNPs and hNPTD transplanted groups after microbead injections. RGC density and distribution in microbead-injected eyes transplanted with hNPIGF-TD cells (E) were similar to the control group (A). (F) Quantification of RGC density in various experimental groups. Data are presented as the fitted geometric means and the 95% confidence limits. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. P < 0.05 compared to NO group; #, P < 0.05 compared to IGF-TD group. Scale bar: 50 μm in A—E. Abbreviations: IGF-TD, transplanted hNPIGF-TD cells after microbead injection; TD, transplanted hNPTD cells after microbead injection. hNP, transplanted untransfected hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection.
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pone.0125695.g005: Quantification of RGC density using ß-III tubulin (red fluorescence) in retinal flatmounts under different experimental conditions.(A—E) Confocal images show significant and robust RGC loss is visualized in the saline, hNPs and hNPTD transplanted groups after microbead injections. RGC density and distribution in microbead-injected eyes transplanted with hNPIGF-TD cells (E) were similar to the control group (A). (F) Quantification of RGC density in various experimental groups. Data are presented as the fitted geometric means and the 95% confidence limits. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. P < 0.05 compared to NO group; #, P < 0.05 compared to IGF-TD group. Scale bar: 50 μm in A—E. Abbreviations: IGF-TD, transplanted hNPIGF-TD cells after microbead injection; TD, transplanted hNPTD cells after microbead injection. hNP, transplanted untransfected hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection.

Mentions: To quantitatively assess the neuroprotective effect of cell transplants, RGCs were counted in retinal flatmounts after immunostaining with ß-III tubulin. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. Data are presented as the fitted geometric means and the 95% confidence limits. Panel A in Fig 5 shows RGC distribution in retinal flatmounts of saline into both anterior chambers and vitreous cavity (Group 1, no glaucoma). In this control group, the RGC density estimated at 5352 (95% confidence limits [CL] of 4214 to 6797) RGCs/mm2. In mice that received microbead injections to their anterior chambers and saline in their vitreous (glaucoma group, SA in Fig 5F), retinal flatmounts showed an approximate 60% reduction in RGC count, with an estimated density of 3196 (95% CL: 2505–4077) RGCs/mm2. This ~60% loss of RGCs corresponds to the expected response to elevated IOP (Fig 5B and SA in panel F). Microbead-injected mice that received untransfected hNPs or hNPTD also exhibited ~60% reductions in RGC density (Fig 5F). The estimated densities for these groups were 2965 (95% CL: 2335–3764) and 3173 (95% CL: 2500–4028) RGCs/mm2, respectively (Fig 5C and 5D, and hNPTD in panel F). Microbead-injected mice received hNPIGF-TD exhibited RGC densities of 5293 with 95% CL of 4168 to 6722 RGCs/mm2 (Fig 5E and IGF in Fig 5F), the latter being not statistically different from saline-injected control group (SA in Fig 5F; P > 0.05). RGC density measurements among microbead-injected eyes for saline, hNPs or hNPTD injected groups were quite similar (all P > 0.05) but statistically different from saline and hNPIGF-TD injected groups (Fig 5F). These data indicate that IGF completely restores IOP-induced RGC loss, as IGF-treated animals had ~99% of RGC density observed in the saline injected controls.


Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival.

Ma J, Guo C, Guo C, Sun Y, Liao T, Beattie U, López FJ, Chen DF, Lashkari K - PLoS ONE (2015)

Quantification of RGC density using ß-III tubulin (red fluorescence) in retinal flatmounts under different experimental conditions.(A—E) Confocal images show significant and robust RGC loss is visualized in the saline, hNPs and hNPTD transplanted groups after microbead injections. RGC density and distribution in microbead-injected eyes transplanted with hNPIGF-TD cells (E) were similar to the control group (A). (F) Quantification of RGC density in various experimental groups. Data are presented as the fitted geometric means and the 95% confidence limits. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. P < 0.05 compared to NO group; #, P < 0.05 compared to IGF-TD group. Scale bar: 50 μm in A—E. Abbreviations: IGF-TD, transplanted hNPIGF-TD cells after microbead injection; TD, transplanted hNPTD cells after microbead injection. hNP, transplanted untransfected hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414591&req=5

pone.0125695.g005: Quantification of RGC density using ß-III tubulin (red fluorescence) in retinal flatmounts under different experimental conditions.(A—E) Confocal images show significant and robust RGC loss is visualized in the saline, hNPs and hNPTD transplanted groups after microbead injections. RGC density and distribution in microbead-injected eyes transplanted with hNPIGF-TD cells (E) were similar to the control group (A). (F) Quantification of RGC density in various experimental groups. Data are presented as the fitted geometric means and the 95% confidence limits. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. P < 0.05 compared to NO group; #, P < 0.05 compared to IGF-TD group. Scale bar: 50 μm in A—E. Abbreviations: IGF-TD, transplanted hNPIGF-TD cells after microbead injection; TD, transplanted hNPTD cells after microbead injection. hNP, transplanted untransfected hNPs after microbead injection; SA, intravitreal saline (no cells) injection after microbead injection; NO, intravitreal saline injection and saline injection into the anterior chamber (no microbead and cell injection). High IOP, elevated intraocular pressure by microbead injection.
Mentions: To quantitatively assess the neuroprotective effect of cell transplants, RGCs were counted in retinal flatmounts after immunostaining with ß-III tubulin. Analysis was conducted using a mixed effects model with a random intercept for each subject followed by the Tukey test on the logarithm of the data. Data are presented as the fitted geometric means and the 95% confidence limits. Panel A in Fig 5 shows RGC distribution in retinal flatmounts of saline into both anterior chambers and vitreous cavity (Group 1, no glaucoma). In this control group, the RGC density estimated at 5352 (95% confidence limits [CL] of 4214 to 6797) RGCs/mm2. In mice that received microbead injections to their anterior chambers and saline in their vitreous (glaucoma group, SA in Fig 5F), retinal flatmounts showed an approximate 60% reduction in RGC count, with an estimated density of 3196 (95% CL: 2505–4077) RGCs/mm2. This ~60% loss of RGCs corresponds to the expected response to elevated IOP (Fig 5B and SA in panel F). Microbead-injected mice that received untransfected hNPs or hNPTD also exhibited ~60% reductions in RGC density (Fig 5F). The estimated densities for these groups were 2965 (95% CL: 2335–3764) and 3173 (95% CL: 2500–4028) RGCs/mm2, respectively (Fig 5C and 5D, and hNPTD in panel F). Microbead-injected mice received hNPIGF-TD exhibited RGC densities of 5293 with 95% CL of 4168 to 6722 RGCs/mm2 (Fig 5E and IGF in Fig 5F), the latter being not statistically different from saline-injected control group (SA in Fig 5F; P > 0.05). RGC density measurements among microbead-injected eyes for saline, hNPs or hNPTD injected groups were quite similar (all P > 0.05) but statistically different from saline and hNPIGF-TD injected groups (Fig 5F). These data indicate that IGF completely restores IOP-induced RGC loss, as IGF-treated animals had ~99% of RGC density observed in the saline injected controls.

Bottom Line: Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects.In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss.RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, 02114, MA, United States of America.

ABSTRACT
We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.

Show MeSH
Related in: MedlinePlus