Transplantation Efficacy of Human Ciliary Epithelium Cells from Fetal Eye and Lin-ve Stem Cells from Umbilical Cord Blood in the Murine Retinal Degeneration Model of Laser Injury

Abstract

A number of degenerative conditions affecting the neural retina including age-related macular degeneration have no successful treatment, resulting in partial or complete vision loss. There are a number of stem cell replacement strategies for recovery of retinal damage using cells from variable sources. However, literature is still deficit in the comparison of efficacy of types of stem cells. The purpose of the study was to compare the therapeutic efficacy of undifferentiated cells, i.e., lineage negative stem cells (Lin-ve SC) with differentiated neurosphere derived from ciliary epithelium (CE) cells on retinal markers associated with laser-induced retinal injury. Laser-induced photocoagulation was carried out to disrupt Bruch's membrane and retinal pigmented epithelium in C57BL/6 mouse model. Lineage negative cells were isolated from human umbilical cord blood, whereas neurospheres were derived from CE of post-aborted human eyeballs. The cells were then transplanted into subretinal space to study their effect on injury. Markers of neurotropic factors, retina, apoptosis, and proliferation were analyzed after injury and transplantation. mRNA expression was also analyzed by real-time polymerase chain reaction at 1 week, and 3-month immunohistochemistry was evaluated at 1-week time point. CE cell transplantation showed enhanced differentiation of rods and retinal glial cells. However, Lin-ve cells exerted paracrine-dependent modulation of neurotrophic factors, which is possibly mediated by antiapoptotic and proliferative effects. In conclusion, CE transplantation showed superior regenerative outcome in comparison to Lin-ve SC for rescue of artificially injured rodent retinal cells. It is imperative that this source for transplantation may be extensively studied in various doses and additional retinal degeneration models for prospective clinical applications.

Keywords: ciliary epithelium cells; laser injury; lineage negative stem cells; retinal degeneration; subretinal; umbilical cord blood.

Figure 1.

Differentiated CE cells transplantation enhances rod cells expression. (A) Immunohistochemistry of rhodopsin showing Cy3 (red fluorescence marked by arrows) expression bound to primary antibody at 20× visualized under confocal microscope in normal control, laser injured retina mice, laser injured transplanted with Lin-ve SC mice, and laser injured transplanted with CE cells mice. (B) Quantitative protein expression of rhodopsin at 1-week time point measured by corrected total cell fluorescence of immunohistochemistry images using Image J software. (C) mRNA expression of rhodopsin in all four groups was analyzed using real-time PCR at 1 week and 3 months after transplantation. Statistical analysis was performed using one-way ANOVA test for immunohistochemistry and real-time PCR results. This was followed by post-hoc analysis using LSD, Scheffe, and Dunnett’s test. *P ≤ 0.05 and **P ≤ 0.01 were regarded as statistically significant. ANOVA: analysis of variance; CE: ciliary epithelium; Lin-ve SC: lineage negative stem cells; PCR: polymerase chain reaction.

Figure 2.

Differentiated CE cell transplantation enhances expression of retinal ganglion cells. (A) Immunohistochemistry of Thy1 showing Cy3 (red fluorescence marked by arrows) expression bound to primary antibody at 20× visualized under confocal microscope in normal control mice, laser injured retina mice, laser injured transplanted with Lin-ve SC mice, and laser injured transplanted with CE cells mice. (B) Quantitative protein expression of Thy1 at 1 week after transplantation was measured by corrected total cell fluorescence of immunohistochemistry images using Image J software (C). mRNA expression of Thy1 in all four groups was analyzed using real-time PCR at 1 week as well as 3 months after transplantation. Statistical analysis was performed using one-way ANOVA test for immunohistochemistry and real-time PCR results. This was followed by post hoc analysis using LSD, Scheffe, and Dunnett’s test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 were regarded as statistically significant. ANOVA: analysis of variance; CE: ciliary epithelium; Lin-ve SC: lineage negative stem cells; PCR: polymerase chain reaction.

Figure 3.

Neurotrophic factor modulation by the Lin-ve SC transplantation. (A) Immunohistochemistry of BDNF showing Cy3 (red fluorescence marked by arrows) expression bound to primary antibody at 20× visualized under confocal microscope in normal control, laser injured mice transplanted with Lin-ve SC, and laser injured mice transplanted with CE cells. (B) Quantitative protein expression of BDNF at 1-week time point measured by corrected total cell fluorescence of immunohistochemistry images using Image J software. (C) mRNA expression of BDNF in all four groups was analyzed using real-time PCR at 1-week as well as 3-month time point. Statistical analysis was performed using one-way ANOVA test for immunohistochemistry and real-time PCR results. This was followed by post hoc analysis using LSD, Scheffe, and Dunnett’s test. *P ≤ 0.05 was regarded as statistically significant. ANOVA: analysis of variance; BDNF: brain-derived neurotrophic factor; CE: ciliary epithelium; Lin-ve SC: lineage negative stem cells; PCR: polymerase chain reaction.

Figure 4.

CE cell transplantation upregulated ciliary neurotrophic factor in the laser injured retina. (A) Immunohistochemistry of CNTF showing Cy3 (red fluorescence marked by arrows) expression bound to primary antibody at 20× visualized under confocal microscope in normal control, laser injured mice, laser injured mice transplanted with Lin-ve SC, and laser injured mice transplanted with CE cells. (B) Quantitative protein expression of CNTF at 1-week time after transplantation measured by corrected total cell fluorescence of immunohistochemistry images using Image J software. (C) mRNA expression of CNTF in all four groups was analyzed using real-time PCR at 1 week as well as 3 months after transplantation. Statistical analysis was performed using one-way ANOVA for immunohistochemistry and real-time PCR results. This was followed by post hoc analysis using LSD, Scheffe, and Dunnett’s test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 were regarded as statistically significant. ANOVA: analysis of variance; CE: ciliary epithelium; CNTF: ciliary neurotrophic factor; Lin-ve SC: lineage negative stem cells; PCR: polymerase chain reaction.

Figure 5.

Antiapoptotic and proliferative activity enhanced by Lin-ve SC transplantation in laser injured retina. (A, B) mRNA expression of antiapoptotic marker Bcl2 and proliferative marker, i.e., Ki67 in all four groups was analyzed using real-time PCR at 1 week as well as 3 months after transplantation. Statistical analysis was performed using one-way ANOVA for real-time PCR results. This was followed by post hoc analysis using LSD, Scheffe, and Dunnett’s test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 were regarded as statistically significant. ANOVA: analysis of variance; CE: ciliary epithelium; Lin-ve SC: lineage negative stem cells; PCR: polymerase chain reaction.