Classification, location, and intensity of granules in retinal pigment epithelium following sodium iodate injection in rat animal model.

Objectives: Age-related macular degeneration (AMD) is one of the eye diseases that can affect a person's central vision. Retinal pigment epithelium (RPE) cells are damaged in this medical condition and some pigments are presented in these cells. Here, we aimed to investigate melanin and lipofuscin granules of RPE cells as a precursor of AMD. Materials and Methods: Hooded rats (n=18) were divided into two groups and received 100 µl of sodium iodate (SI) into the retro-orbital sinus of their eyes at 40 and 60 mg/kg doses. The total number of melanin and lipofuscin granules, different types of granules, cytoplasmic dispersion of granules as well as morphological changes in the shape and number of nuclei of RPE cells were evaluated over the course of 1-30 days. Results: The total number of melanin pigments increases over time at a dose of 40 mg/kg and decreases at a dose of 60 mg/kg. Also, the total number of lipofuscin granules in 40 mg/kg increases over time and decreases in 60 mg/kg. Autofluorescent intensity (AF) is also increased at 40 mg/kg, but at 60 mg/kg, the highest intensity is on day 7. Also, the highest number of multinucleated giant cells was on day 7 at 60 mg/kg and the most changes in cell appearance due to sodium iodate injection were seen on the first day after injection. Conclusion: We demonstrated that granules and autofluorescent intensity appear to decrease at high doses of sodium iodate, which is similar to the advanced stage of the AMD disease, where the number of granules and AF intensity increase in the middle and even early stages of the disease.

Generation of Retinal Pigmented Epithelium-Like Cells from Pigmented Spheres Differentiated from Bone Marrow Stromal Cell-Derived Neurospheres.

Background: Retinal degeneration causes blindness, and cell replacement is a potential therapy. The purpose of this study is to formation of pigmented neurospheres in a simple medium, low-cost, high-performance manner over a short period of time while expressing markers of RPE cells and the activation of specific genes of the pigment cells. Also, these neurospheres have the ability to produce a monolayer of retinal pigment epithelium-like cells (RPELC) with the ability of photoreceptor outer segment phagocytosis. Methods: BMSC were isolated from pigmented hooded male rats and were immunoreactive to BMSC markers, then converted into neurospheres, differentiated into pigmented spheres (PS), and characterized using Retinal pigment epithelium-specific 65 kDa protein (RPE65), Retinaldehyde-binding protein 1 (CRALBP) and orthodenticle homeobox 2 (OTX2) markers by immunocytochemistry, RT-PCR and RT-qPCR. The PS were harvested into RPELC. The functionality of RPELC was evaluated by phagocytosis of fluorescein-labeled photoreceptor outer segment. Results: The BMSC immunophenotype was confirmed by immunostained for fibronectin, CD90, CD166 and CD44. These cells differentiated into osteogenic and lipogenic cells. The generated neurospheres were immunoreactive to nestin and stemness genes. The PS after 7-14 days were positive for RPE65 (92.76-100%), CRALBP (95.21-100%) and OTX2 (94.88-100%), and after 30 days RT-PCR, qPCR revealed increasing in gene expression. The PS formed a single layer of RPELC after cultivation and phagocyte photoreceptor outer segments. Conclusion: Bone marrow stromal stem cells can differentiate into functional retinal pigmented epithelium cells in a simple, low-cost, high-performance manner over a short period of time. These cells due to expressing the RPELC genes and markers can be used in cell replacement therapy for degenerative diseases including age-related macular degeneration as well as retinitis pigmentosa.

High efficient differentiation of human adipose-derived stem cells into retinal pigment epithelium-like cells in medium containing small molecules inducers with a simple method.

BACKGROUND: The induction of retinal pigmented epithelium cells (RPE) is one of the most important objectives in research focused on treating retinal degenerative diseases. The present study aims to differentiate human adipose stem cells (hADSCs) into RPE cells for replacement therapies in cases of retinal degenerative diseases. METHODS: Lipoaspirate-derived human adipose stem cells (LA-hADSCs) were obtained from abdominal samples and examined by immunocytochemistry for the expression of mesenchymal adipose stem cell markers. RPE cells were also obtained from human samples and cultured to be used as control after being examined for the expression of their designated markers. hADSCs differentiated into RPE cells after 80 days using chemical inducers in one steps. The differentiated cells were then compared to control cells in marker expression. The differentiated cells were also examined under a scanning electron microscope for the presence of apical microvilli and cell connection. RESULTS: Cultured hADSCs at the fourth passage was shown to express the surface markers CD90 (98 ± 2%), CD11b (96 ± 3%), and CD105 (95 ± 4%). The RPE cells obtained from human samples expressed the marker RPE65 quite well. 80 days after differentiation, the previously hADSCs expressed both RPE65 (100%) and CRALBP (96 ± 1%) and were thus significantly similar to the RPE cells obtained from human samples. Morphologically, differentiated cells appeared to have epithelial and cytoplasmic pigment granules. Observations using a scanning electron microscope recorded clear connections among the differentiated RPE cells and revealed apical microvilli. CONCLUSION: Human adipose stem cells can differentiate into retinal pigmented epithelium cells, which can be used in cell replacement therapy for degenerative diseases including age-related macular degeneration (AMD) as well as retinitis pigmentosa (RP).

Survival and Migration of Adipose-Derived Stem Cells Transplanted in the Injured Retina.

OBJECTIVES: Transplantation of stem cells is one of the approaches to treat retinal diseases. Our objective was to determine whether adipose-derived stem cell transplant can survive and migrate in the injured retina using a sodium iodate model for the pigmented retinal epithelium injury. MATERIALS AND METHODS: The adipose-derived stem cells were isolated from male albino Sprague-Dawley rats and labeled with DiI so as to track the transplants in the subretinal space. Retinal pigmented epithelium damage was induced by retro-orbital sinus sodium iodate injection (40 mg/kg) into albino Sprague-Dawley rats. Four weeks after transplantation, the eyeballs were fixed in 4% paraformaldehyde and cut with cryostat. The eyeballs were serially sectioned along the vertical meridian. Cryosections were from the full length of the retina and passing through the optic nerve head. The survival and migration of transplanted cells were assessed. RESULTS: Sodium iodate selectively destroyed the retinal pigmented epithelium layer. The transplanted cells incorporated into the retinal pigmented epithelium layer, perhaps differentiating into a retinal pigmented epithelium phenotype. The transplanted cells were located in the subretinal space; after 4 weeks, some were observed in the retinal pigmented epithelium layer. CONCLUSIONS: We found that adipose-derived stem cells survived for 4 weeks after transplantation and migrated into the retinal pigmented epithelium layer.