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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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Detection of tdTomato (TD) and IGF1-TD fusion protein in human neuronal progenitor cells (hNP).(A) Expression of TD protein in transfected hNPs by their red fluorescence. (B) TD protein can also be detected by using immunohistochemistry (FITC, green). (C) Expression of IGF-TD fusion protein in hNPs by their red fluorescence. (D) Detection the IGF-1 moiety of the IGF-TD fusion protein in transfected cells by immunostaining (FITC, green). (E) Expression of IGF-1 component of the IGF-TD mRNA in hNPIGF-TD cells by qRT-PCR; IGF-1 mRNA is undetectable in untransfected hNP and hNPTD cells. (F) Western blot analysis of IGF-1 component of the IGF-TD protein in hNPIGF-TD cell lysates confirms the expected molecular weight of approximately 60 kD. IGF-1 was not detected in untransfected hNP and hNPTD cells. (G) IGF-1 levels are higher in the medium of cells transfected with the IGF-TD fusion protein (red triangles). Data are presented as mean ± SE for the day 0 wells (B; blank wells). Symbols correspond to the fitted means ± the 95% confidence limits of the measurements conducted in conditioned medium from the difference hNPs (open symbols; empty vector, yellow symbols; TD-transfected cells, green symbols; IGF-TD—transfected cells). Analysis was conducted by fitting a two way linear model for treatments and time points on the inverse of the data. Multiple comparisons were conducted on the transformed scale by the Tukey test. Asterisks indicate statistically significant differences versus all groups at the same time point. Abbreviations: hNP, neuronal progenitor cells; TD, tdTomato; IGF-TD, IGF-1-tdTomato. Scale bar in A—D: 50 μm.
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pone.0125695.g001: Detection of tdTomato (TD) and IGF1-TD fusion protein in human neuronal progenitor cells (hNP).(A) Expression of TD protein in transfected hNPs by their red fluorescence. (B) TD protein can also be detected by using immunohistochemistry (FITC, green). (C) Expression of IGF-TD fusion protein in hNPs by their red fluorescence. (D) Detection the IGF-1 moiety of the IGF-TD fusion protein in transfected cells by immunostaining (FITC, green). (E) Expression of IGF-1 component of the IGF-TD mRNA in hNPIGF-TD cells by qRT-PCR; IGF-1 mRNA is undetectable in untransfected hNP and hNPTD cells. (F) Western blot analysis of IGF-1 component of the IGF-TD protein in hNPIGF-TD cell lysates confirms the expected molecular weight of approximately 60 kD. IGF-1 was not detected in untransfected hNP and hNPTD cells. (G) IGF-1 levels are higher in the medium of cells transfected with the IGF-TD fusion protein (red triangles). Data are presented as mean ± SE for the day 0 wells (B; blank wells). Symbols correspond to the fitted means ± the 95% confidence limits of the measurements conducted in conditioned medium from the difference hNPs (open symbols; empty vector, yellow symbols; TD-transfected cells, green symbols; IGF-TD—transfected cells). Analysis was conducted by fitting a two way linear model for treatments and time points on the inverse of the data. Multiple comparisons were conducted on the transformed scale by the Tukey test. Asterisks indicate statistically significant differences versus all groups at the same time point. Abbreviations: hNP, neuronal progenitor cells; TD, tdTomato; IGF-TD, IGF-1-tdTomato. Scale bar in A—D: 50 μm.

Mentions: hNPIGF-TD cells were generated by transfecting them with a vector containing the coding sequences of IGF-TD, in which the TD sequence was tagged to the C-terminus of IGF-1 producing a fusion protein. Similarly, hNPTD cells were generated by inserting the TD coding sequence alone. Immunostaining of transfected cells with antibodies against TD or IGF-1 confirmed the expression of TD and IGF-TD proteins in specific cells (Fig 1A–1D). High levels of mouse IGF-1 mRNA was also detected by real time RT-PCR in hNPIGF-TD cells but not in hNPTD or untransfected hNP cells (Fig 1E). The synthesis and secretion of IGF-TD protein by hNPIGF-TD cells were detected from the cell lysates using Western blot analysis (Fig 1F). ELISA was also used to evaluate the concentration of IGF-TD in the conditioned media collected from cultured hNPIGF-TD and hNPTD cells at 1 and 3 days post-transfection. The concentration of IGF-TD protein gradually increased to about 0.5 ng/ml on day 3 (Fig 1G). In contrast, there were very low levels of IGF-1 in hNPTD or hNPs (Fig 1G).


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)

Detection of tdTomato (TD) and IGF1-TD fusion protein in human neuronal progenitor cells (hNP).(A) Expression of TD protein in transfected hNPs by their red fluorescence. (B) TD protein can also be detected by using immunohistochemistry (FITC, green). (C) Expression of IGF-TD fusion protein in hNPs by their red fluorescence. (D) Detection the IGF-1 moiety of the IGF-TD fusion protein in transfected cells by immunostaining (FITC, green). (E) Expression of IGF-1 component of the IGF-TD mRNA in hNPIGF-TD cells by qRT-PCR; IGF-1 mRNA is undetectable in untransfected hNP and hNPTD cells. (F) Western blot analysis of IGF-1 component of the IGF-TD protein in hNPIGF-TD cell lysates confirms the expected molecular weight of approximately 60 kD. IGF-1 was not detected in untransfected hNP and hNPTD cells. (G) IGF-1 levels are higher in the medium of cells transfected with the IGF-TD fusion protein (red triangles). Data are presented as mean ± SE for the day 0 wells (B; blank wells). Symbols correspond to the fitted means ± the 95% confidence limits of the measurements conducted in conditioned medium from the difference hNPs (open symbols; empty vector, yellow symbols; TD-transfected cells, green symbols; IGF-TD—transfected cells). Analysis was conducted by fitting a two way linear model for treatments and time points on the inverse of the data. Multiple comparisons were conducted on the transformed scale by the Tukey test. Asterisks indicate statistically significant differences versus all groups at the same time point. Abbreviations: hNP, neuronal progenitor cells; TD, tdTomato; IGF-TD, IGF-1-tdTomato. Scale bar in A—D: 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0125695.g001: Detection of tdTomato (TD) and IGF1-TD fusion protein in human neuronal progenitor cells (hNP).(A) Expression of TD protein in transfected hNPs by their red fluorescence. (B) TD protein can also be detected by using immunohistochemistry (FITC, green). (C) Expression of IGF-TD fusion protein in hNPs by their red fluorescence. (D) Detection the IGF-1 moiety of the IGF-TD fusion protein in transfected cells by immunostaining (FITC, green). (E) Expression of IGF-1 component of the IGF-TD mRNA in hNPIGF-TD cells by qRT-PCR; IGF-1 mRNA is undetectable in untransfected hNP and hNPTD cells. (F) Western blot analysis of IGF-1 component of the IGF-TD protein in hNPIGF-TD cell lysates confirms the expected molecular weight of approximately 60 kD. IGF-1 was not detected in untransfected hNP and hNPTD cells. (G) IGF-1 levels are higher in the medium of cells transfected with the IGF-TD fusion protein (red triangles). Data are presented as mean ± SE for the day 0 wells (B; blank wells). Symbols correspond to the fitted means ± the 95% confidence limits of the measurements conducted in conditioned medium from the difference hNPs (open symbols; empty vector, yellow symbols; TD-transfected cells, green symbols; IGF-TD—transfected cells). Analysis was conducted by fitting a two way linear model for treatments and time points on the inverse of the data. Multiple comparisons were conducted on the transformed scale by the Tukey test. Asterisks indicate statistically significant differences versus all groups at the same time point. Abbreviations: hNP, neuronal progenitor cells; TD, tdTomato; IGF-TD, IGF-1-tdTomato. Scale bar in A—D: 50 μm.
Mentions: hNPIGF-TD cells were generated by transfecting them with a vector containing the coding sequences of IGF-TD, in which the TD sequence was tagged to the C-terminus of IGF-1 producing a fusion protein. Similarly, hNPTD cells were generated by inserting the TD coding sequence alone. Immunostaining of transfected cells with antibodies against TD or IGF-1 confirmed the expression of TD and IGF-TD proteins in specific cells (Fig 1A–1D). High levels of mouse IGF-1 mRNA was also detected by real time RT-PCR in hNPIGF-TD cells but not in hNPTD or untransfected hNP cells (Fig 1E). The synthesis and secretion of IGF-TD protein by hNPIGF-TD cells were detected from the cell lysates using Western blot analysis (Fig 1F). ELISA was also used to evaluate the concentration of IGF-TD in the conditioned media collected from cultured hNPIGF-TD and hNPTD cells at 1 and 3 days post-transfection. The concentration of IGF-TD protein gradually increased to about 0.5 ng/ml on day 3 (Fig 1G). In contrast, there were very low levels of IGF-1 in hNPTD or hNPs (Fig 1G).

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