Neural differentiation of human retinal pigment epithelial cells on alginate/gelatin substrate.

Purpose: The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments and specific growth factors motivate the differentiation of human retinal pigment epithelial (hRPE) cells toward a retinal neural lineage. In this study, we evaluated alginate/gelatin (A/G) as a substrate for the culture of hRPE cells. Methods: hRPE cells were isolated from neonatal human cadaver globes and cultivated on A/G substrate under different culture conditions, including 30% human amniotic fluid (HAF), 10% fetal bovine serum (FBS), and serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). The proliferation of cells in different culture conditions was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a cell proliferation assay. Immunocytochemistry and real-time PCR were performed to evaluate the effect of the substrate on hRPE cell differentiation. Results: A significant increase in the cell proliferation rate was observed in hRPE cells cultivated on an A/G substrate. Continuous observations demonstrated that hRPE cells formed densely packed, suspended spheroids in DMEM/F12 culture conditions, with dominant transdifferentiation into amacrine cells. Small adherent clusters of hRPE cells in HAF- and FBS-treated cultures represented dedifferentiation toward retinal progenitor cells. These cultures generated amacrine, rod photoreceptors, and bipolar cells. Conclusions: These findings indicated that A/G substrate induced neural retinal cell propagation in cultures and would therefore be promising for RPE-based tissue engineering studies.

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

PURPOSE: Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. METHODS: RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. RESULTS: Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. CONCLUSIONS: This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.