hRPE Cell Research Articles

Grafting of ARPE-19 and Schwann Cells to the Subretinal Space in RCS Rats

To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue. Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells. Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time. Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.

The bovine vitreous-derived lipid factor (bVLF) is a powerful inhibitor of retinal pigmented epithelial (hRPE) cell proliferation

Human retinal pigmented epithelial cell (hRPE) proliferation plays a significant role in various proliferative diseases associated to the retina that leads to loss of vision, such as proliferative vitreoretinopathy. In the current study, the role of the bovine vitreous lipid factor (bVLF) in hRPE cell proliferation has been investigated. bVLF is a bioactive lipid isolated from the bovine vitreous body with strong Ca 2+-mobilizing activity in fibroblast. In the first approach, the effects of bVLF on Ca 2+-mobilizing activity were investigated in hRPE. The results showed that bVLF induced, in a dose-dependent manner, a Ca 2+ mobilization from PA-sensitive intracellular stores [non-Ins(1,4,5)P 3-sensitive stores], in which extracellular Ca 2+ participated. The increase in intracellular Ca 2+ was associated with a dose-dependent inhibiting effect on cell proliferation. At a dose of 10 μg/mL, bVLF caused a 26% or a 44% inhibition in hRPE cell proliferation during the 3- or the 6-day culture periods, respectively. These effects appear to be specific in hRPE cells, since EFGR-T17 fibroblast cells treated with equivalent amounts of bVLF did not show any inhibiting effects. This inhibitory action was not associated to apoptotic/necrotic processes. Furthermore, bVLF inhibited EGF-, bFGF-, IGF-I-, PDGF-, HGF- and VEGF-induced proliferation of the hRPE cells. Moreover, this inhibitory response was also observed in FBS-induced hRPE cell proliferation. bVLF, at a concentration of 10 μg/mL, induced 16% inhibition of proliferation during a culture period of 3 days. This inhibitory action was greater during the 6-day culture period, exceeding 40%. With regard to this action, the results showed that bVLF has a potent inhibitory effect on ERK1/2 activation, and plays a key role in the control of hRPE cell proliferation. These observations contribute to the knowledge of inhibitory factors responsible for keeping antiproliferative environment that preserve the RPE-associated activities in normal states. It advances the interesting possibility that this factor or a factor with characteristics common to bVLF might be involved in the pathogenesis of abnormal proliferative eye processes.

Amniotic membrane as support for human retinal pigment epithelium (RPE) cell growth.

The aim of this work was to culture human retinal pigment epithelium (hRPE) cells over human amniotic membrane (hAM). Human AM was studied for its viability as an adequate support for transplantation of an hRPE cell monolayer with preserved cell polarity to the subretinal space. Human AM was obtained from pregnant women during caesarean section. The hAM was sectioned and the pieces were fixed to culture dishes. Human RPE cells were cultured from adult corneal donors and were seeded over hAM. Phase-contrast photographs were obtained. Selected specimens were processed by transmission electronic microscopy (TEM). The attachment and growth of hRPE cells over hAM was observed. Human RPE cells constituted tight colonies that maintained epithelial phenotype. Using TEM, we identified a monolayer of hRPE cells, with cuboidal to spheroidal morphology. These cells showed integration with the substrate and cell-cell contacts were detected. Amniotic membrane may be a suitable substrate for hRPE growth. Further studies are required in order to determine the viability of hRPE on hAM in the subretinal space.

A c-mycAntisense Oligonucleotide Inhibits Human Retinal Pigment Epithelial Cell Proliferation

The purpose of this work was to investigate if MYC-dependent intracellular mitogenic pathway is active in cultures of human retinal pigment epithelial (hRPE) cells and whethermycantisense phosphorotioate oligonucleotides (c-myc-AS-ODN) are useful tools for inhibiting the proliferation of hRPE cells. Cultures of hRPE cells were established from adult human corneal donors. These cells were positively stained for cytokeratins and vimentin.MycmRNA expression was determined by Northern blot analysis and it was determined by means of immunofluorescence if MYC was expressed.C-myc-AS-ODN effect on cell proliferation was estimated by evaluating the incorporation of 5-bromo-2′-deoxy-uridine into cellular DNA. Cell number was estimated by using a tetrazolium bromide based colorimetric method. Human RPE cells in culture expressed MYC andmycmRNA as well as prothymosin α mRNA – a gene whose transcription is under MYC control – indicating that MYC-dependent intracellular mitogenic pathway is active in these cells. In accordance with this, we found that blocking the expression ofmycby the addition of c-myc-AS-ODN to the culture medium inhibited hRPE cell proliferation. The effect of the c-myc-AS-ODN was found to be sequence specific (the use of a control oligonucleotide with the same sequence but in an opposite direction had no effect) and dose-dependent (4 μmwas the lowest effective dose tested). By using RT-PCR we found that the c-myc-AS-ODN inhibition of cell proliferation was related to a diminution in c-mycmRNA expression, and by immunofluorescence we detected a diminution in c-MYC protein staining in RPE cells after 48 hr of treatment with c-myc-AS-ODN. Furthermore, growth inhibition remained for at least 5 days after addition of a single dose of the c-myc-AS-ODN to the culture. We conclude that hRPE cell proliferation is under MYC control. Blocking the expression ofmycby c-myc-AS-ODN inhibited hRPE cell proliferation. These findings establish a rationale for investigating the potential use of a c-myc-AS-ODN as a novel therapeutical tool in the treatment of Proliferative Vitreoretinopathy.

Human retinal pigment epithelial cells from different donors continuously produce a vascular endothelial cell-stimulating factor into serum-free medium

Mitogenic activities of human retinal pigment epithelial cell-conditioned medium (HRPE-CM) with different effects, such as inhibition, stimulation or no effect, on the proliferation of vascular endothelial cells (EC) in vitro have been reported. In this study, 14 HRPE cell lines were established from normal human eyes. Human umbilical vein endothelial cells (HUVEC) in the early passages were used as target cells to detect the mitogenic activity of HRPE-CM on the growth of vascular EC. Our results confirm that HRPE cells in culture continuously synthesize and secrete HUVEC growth substance(s) into a serum-free medium. The ability of HRPE cell lines to produce this mitogen seem unrelated either to in vivo donor factors or to in vitro cell life span. Using an enzyme-linked immunosorbance assay, we demonstrated that only HRPE cell extract, not HRPE-CM, can be recognized by basic fibroblast growth factor (bFGF)-specific antibody, though identical bioactivities on the growth of HUVEC were found in both preparations. The active component in HRPE-CM was heat- and trypsin-sensitive, and stable at extremes of pH (2.5 to 10.0). In addition, the bioactive molecule could not pass through a M(r) 30,000 cut-off membrane, suggesting that it is a fairly high molecular mass polypeptide. These observations suggest that the EC growth factor in HRPE-CM is distinct from fibroblast growth factors (FGFs).