Human Fetal Retinal Pigment Epithelium Research Articles

886. AAV-Mediated Somatic Gene Transfer as an Approach To Delineate Pathogenic Mechanisms in an Autosomal Dominant Blindness Disorder Resembling Age-Related Macular Degeneration (AMD)

Malattia Leventinese (ML) is an autosomal dominant inherited macular degeneration with a middle-age onset. Phenotypically, the disease closely resembles Age-related Macular Degeneration (AMD), a condition affecting 20% of the population over age 65 and the leading cause of blindness in the elderly in the Western world. Patients with ML suffer from decreased visual acuity and have a characteristic radial pattern of deposits, called drusen, which form beneath the retinal pigment epithelium (RPE). A single missense mutation (R345W) in the Epidermal Growth Factor Containing Fibrillin-like Extracellular Matrix Protein 1 (EFEMP1) gene is responsible for the disease, and leads to the misfolding and inefficient secretion of the protein. In normal retinas, the protein localizes to the inner photoreceptor matrix (IPM), apical to the RPE, while in individuals with ML, it resides between the RPE and drusen. We therefore surmised that wild-type EFEMP1 is apically secreted by the RPE, and that the R345W mutation misdirects this secretion basolaterally. To test this hypothesis, we utilized a cell culture system consisting of polarized monolayers of human fetal RPE (hfRPE) cells grown on Transwell membranes. AAV2/1 vectors containing FLAG-tagged wild-type or mutant EFEMP1 were used to transduce the hfRPE cells with high efficiency. Transduction did not affect hfRPE cell polarization or tight junction formation, as shown by immunofluorescence staining for Na+/K+ ATPase, CD147, and ZO-1. Immunoprecipitation of EFEMP1-FLAG from the basolateral and apical medium revealed that, contrary to our hypothesis, EFEMP1 is secreted basolaterally, and that this polarized secretion pattern is not altered by the R345W mutation. In addition, we found that this pattern of secretion was distinct from dog epithelial (MDCK) cells, which secreted the protein apically. Additional immunoprecipitation and immunofluorescence experiments indicated that the R345W mutation resulted in retention of EFEMP1 within polarized RPE cells. We conclude that EFEMP1 is directionally secreted from epithelial cells, and that its basolateral secretion from RPE cells may be indicative of some yet unknown function in the extracellular matrix underlying those cells, within the local environment of drusen formation in ML patients. However, the absence of an alteration in polarized secretion suggests that the intracellular retention of mutant EFEMP1 may be a major contributing factor to the progression of ML.

Derivation and Comparative Assessment of Retinal Pigment Epithelium from Human Embryonic Stem Cells Using Transcriptomics

Human stem-cell derivatives are likely to play an important role in the future of regenerative medicine. Evaluation and comparison to their in vivo counterparts is critical for assessment of their therapeutic potential. Transcriptomics was used to compare a new differentiation derivative of human embryonic stem (hES) cells--retinal pigment epithelium (RPE)--to human fetal RPE. Several hES cell lines were differentiated into putative RPE, which expressed RPEspecific molecular markers and was capable of phagocytosis, an important RPE function. Isolated hES cell-derived RPE was able to transdifferentiate into cells of neuronal lineage and redifferentiate into RPE-like cells through multiple passages (>30 Population doublings). Gene expression profiling demonstrated their higher similarity to primary RPE tissue than of existing human RPE cell lines D407 and ARPE-19, which has been shown to attenuate loss of visual function in animals. This is the first report of the isolation and characterization of putative RPE cells from hES cells, as well as the first application of transcriptomics to assess embryonic stem-cell derivatives and their in vivo counterparts--a "differentiomics" outlook. We describe for the first time, a differentiation system that does not require coculture with animal cells or factors, thus allowing the production of zoonoses-free RPE cells suitable for subretinal transplantation in patients with retinal degenerative diseases. With the further development of therapeutic cloning, or the creation of the banks of homozygous human leucocyte antigen (HLA) hES cells using parthenogenesis, RPE lines could be generated to overcome the problem of immune rejection and could be one of the nearest term applications of stem-cell technology.

Retinal pigment epithelial function: a role for CFTR?

In the vertebrate eye, the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE) are separated by a small extracellular (subretinal) space whose volume and chemical composition varies in the light and dark. Light onset triggers relatively fast (ms) retinal responses and much slower voltage and resistance changes (s to min) at the apical and basolateral membranes of the RPE. Two of these slow RPE responses, the fast oscillation (FO) and the light peak, are measured clinically as part of the electrooculogram (EOG). Both EOG responses are mediated in part by apical and basolateral membranes proteins that form a pathway for the movement of salt and osmotically obliged fluid across the RPE, from retina to choroid. This transport pathway serves to control the volume and chemical composition of the subretinal and choroidal extracellular spaces. In human fetal RPE, we have identified one of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) by RT-PCR, immunolocalization, and electrophysiological techniques. Evidence is presented to suggest that the FO component of the EOG is mediated directly or indirectly by CFTR.

Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection

This paper demonstrates lentiviral transduction of the humanized form of the Aequoria victoria gene for green fluorescent protein (GFP) into human fetal retinal pigment epithelium (RPE) in vitro and rabbit RPE in vivo. In vitro GFP expression of cultured human fetal RPE begins within two to three days after 12–16 h of maintained exposure to the virus at titers of 10 8–10 9 infectious units (IU)/ml. Both stationary and dividing cells are transduced using a lenti viral vector with a cytomegalovirus (CMV) promoter. Expression remains stable for at least three to four months without evidence of toxicity and continues through cell division. In vivo expression is followed non-invasively in rabbit eye using a scanning laser ophthalmoscope (SLO), which can detect single fluorescing retinal cells. In vivo expression begins within a few days after a viral solution is introduced into the subretinal space. A solution of 10 9 IU/ml produces fluorescence within three to four days. Less concentrated solutions lead to slower and less expression. No expression is detectable at concentrations of 10 6 IU/ml. Within one to two weeks after introduction of the viral solution, there is evidence of rejection seen by SLO as a loss of GFP fluorescence and disruption of the RPE. Histology shows damage to the RPE layer and monocytic cell infiltrates in the choroid and subretinal space within the area receiving the viral solution. Strong GFP expression leads to rejection within two weeks. With less expression, rejection is delayed and in some cases undetectable for at least six months. If the GFP gene is not included in the viral vector or if the viral concentration is insufficient to produce detectable GFP expression, rejection is not seen. Using a rhodopsin promoter or injecting the virus intra rather than subretinally produces weak expression and no rejection. Lentivirus can induce expression of a foreign gene in the RPE. Viral induced transduction and GFP expression have no effect on the viability of the RPE in vitro. Continued expression of GFP after cell division implies chromosomal integration of the gene. In vivo expression of GFP in RPE encounters rejection. Rejection may not occur with low GFP expression. The latter occurs with low viral titers, a rhodopsin promoter or intra-retinal injection of viral solution. The results are relevant to gene therapy in retina when gene transduction leads to the expression of foreign proteins.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL

Purpose. To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. Methods. Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-? activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [ 3 H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. Results. HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-? activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-? activated HFRPE cells. Conclusions. Although HFRPE cells express TRAIL and its expression is upregulated by IFN-? activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.

Cytokine modulation of costimulatory molecules on human fetal retinal pigment epithelial cells

Purpose. Experiments were performed to evaluate the effect of various pro- and anti-inflammatory cytokines on the human fetal retinal pigment epithelium's (HFRPE) expression of major histocompatibility complex (MHC) and costimulatory molecules. Methods. Pure cultures of HFRPE cells were isolated. HFRPE cells were incubated in the presence of Interferon-? (IFN-?), IFN-ß, Tumor Necrosis Factor-a (TNF-a), Interleukin-1ß (IL-1ß), Tumor Growth Factor-ß (TGF-ß), and a combination of IFN-? and TGF-ß (pre-incubation and simultaneously incubated). The expression of MHC class I and class II, Intercellular cell adhesion molecule (ICAM-1), B7-1 (CD80), and B7-2 (CD86) molecules was quantitatively analyzed by flow cytometry. Results. The cultured HFRPE cells expressed high levels of MHC class I and low levels of MHC class II and ICAM-1 molecules. After culture with the above mentioned cytokines, IFN-? up-regulated the HFRPE’s expression of MHC class II and ICAM-1. IFN-ß and IL-1ß only up-regulated the expression of ICAM-1. TGF-ß was unable to suppress the up-regulatory effect of IFN-? in HFRPE cells (pre-incubated and simultaneously incubated). The other cytokines did not have any significant effect on HFRPE’s expression of MHC I and II or the selected costimulatory molecules. Conclusions. Our findings indicate that TGF-ß cannot suppress up-regulating effects of IFN-? on HFRPE’s expression of MHC and costimulatory molecules. Overall, the weak or lack of expression of costimulatory molecules after stimulation with various cytokines further confirms that HFRPE cells are weak antigen presenting cells.

A model for xenogenic immune response

To develop a model for analyzing the immune response after xenogenic human fetal retinal pigment epithelium (HFRPE) transplantation. Pure sheets of HFRPE cells were isolated and attached to poly (DL-lactide-co-glycolide) polymer films and HFRPE spheroids were formed. The spheroids were transplanted into the subretinal space of New Zealand albino rabbits and were observed for 5 months. Bare polymer films were transplanted into the subretinal space of Dutch Belted pigmented rabbits, serving as control. The polymer film was biodegraded within 3 weeks in the subretinal space. No signs of inflammation in the retina or choroid were observed. The HFRPE spheroids were easily transplanted into the subretinal space. The immune response was followed with ophthalmoscopy. Light microscopy indicated a localized immune response in the transplanted area in which the retina and the choroid were infiltrated with immune cells. This infiltration was denser in the choroid. HFRPE spheroid transplantation may be utilized as a model for studying the xenogenic immune response after HFRPE transplantation. This model may also have applications in evaluating the role of immune suppressive agents in preventing rejection after HFRPE transplantation.

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

Purpose. The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. Methods. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. Results. Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and lower Bax levels compared to cells grown on uncoated dishes. However, Bcl-X L and c-Fos levels were comparable in the two cultures. After UV-A or DNR treatment, Bcl-2, Bcl-X L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishes. Conclusion. A significant protection against apoptosis of RPE cells grown on ECM compared to cells grown on uncoated plastic dishes was found after exposure to UV-A or DNR. This protection was found to be proportionally correlated to the anti-apoptotic protein Bcl-2 and inversely correlated to the expression of Bax. Furthermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. These findings demonstrate that ECM is of great importance for RPE cell survival during noxious stimuli and points out the essential role for a healthy Bruch’s Membrane (BM) for RPE survival.

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and lower Bax levels compared to cells grown on uncoated dishes. However, Bcl-X L and c-Fos levels were comparable in the two cultures. After UV-A or DNR treatment, Bcl-2, Bcl-X L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishes. A significant protection against apoptosis of RPE cells grown on ECM compared to cells grown on uncoated plastic dishes was found after exposure to UV-A or DNR. This protection was found to be proportionally correlated to the anti-apoptotic protein Bcl-2 and inversely correlated to the expression of Bax. Furthermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. These findings demonstrate that ECM is of great importance for RPE cell survival during noxious stimuli and points out the essential role for a healthy Bruch's Membrane (BM) for RPE survival.

Human fetal retinal pigment epithelium suppresses the activation of CD4(+) and CD8(+) T-cells.

The suppressive effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human CD4(+) and CD8(+) T-cells was evaluated in vitro. Pure populations of CD4(+) and CD8(+) T-cells were isolated from human peripheral blood-derived buffy coats by negative immunomagnetic selection. The purity of the cells was examined by flow cytometry using anti-CD3-FITC, anti-CD4-FITC, anti-CD8-PE, and anti-CD20-PE mAbs. HFRPE cells were isolated from fetal eyes and pure cultures were obtained. The effect of normal or IFN-gamma-activated HFRPE cells at early (P3) or late (P6) passages on the activation of CD4(+) and CD8(+) T-cells was assessed in two different T-cell activation assays. In both activation models anti-CD3 mAb (OKT3) provided the antigen-specific signal. The secondary signal for the activation of CD4(+) and CD8(+) T-cells was provided with anti-CD18 mAb (TS1/18) and anti-CD28 mAb (9.3) in the first and the second assay respectively. Cross-linking of these soluble mAbs was performed with sheep-anti-mouse IgG-coated latex beads. The T-cell activation was determined by cell proliferation measured by [(3)H]thymidine incorporation. In each activation assay T-cells were incubated with HFRPE cells in a ratio of T-cells to HFRPE of 1:1 or 1:4. CD4(+) and CD8(+) T-cells were activated by cross-linking CD3 and CD18 in the first assay (CD3/CD18) and CD3 and CD28 in the second assay (CD3/CD28). In both assays HFRPE inhibited the activation of CD4(+) and CD8(+) T-cells. IFN-gamma-activated HFRPE cells totally suppressed the T-cell activation at a 1:1 ratio. This suppressive effect was weaker at lower cell ratios. Some donor variation was observed in the inhibition at the lower cell ratios, especially for the inhibition of CD8(+) T-cell activation with anti-CD3/CD18. The passaging of HFRPE cells did not alter their suppressive effect on CD4(+) and CD8(+) T-cells. HFRPE cells suppressed the activation of both CD4(+) and CD8(+) T-cells in vitro. These findings suggest that RPE-induced immune suppression may play a significant role in maintaining immune privilege in the subretinal space.

Human fetal retinal pigment epithelial cells induce apoptosis in allogenic T-cells in a Fas Ligand and PGE2 independent pathway

Purpose. To evaluate the inhibitory effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human T-cells. Methods. Pure cultures of HFRPE cells were incubated with purified human T-cells in three different activation assays: 1) allogenic peripheral blood mononuclear cells; 2) OKT3 coated beads in the presence of accessory cells; and 3) stimulation with phorbol ester and phytohemagglutinin. Results. HFRPE cells suppressed the activation of T-cells in all three assays. The mechanism of HFRPE mediated T-cell suppression was apoptosis. The role of Fas ligand(FasL)/Fas-mediated T-cell suppression was excluded, since FasL protein or mRNA could not be detected on HFRPE cells with flow cytometry and by reverse transcriptase polymerase chain reaction, respectively. Additionally, the inhibitory effect of HFRPE cells could not be blocked by anti-Fas ligand or antagonistic anti-Fas antibodies. Moreover, HFRPE cells suppressed the proliferation of anti-CD3 mAb mediated T-cell proliferation of murine splenocytes isolated from lpr mice. The inhibitory effect of HFRPE cells was not PGE 2 mediated, since indomethacin could not restore the T-cell activation. Although the HFRPE mediated T-cell apoptosis was cell-cell contact independent, it was not induced by secretion of TNF-a, TGF-ß, or IL-10. The ratio between HFRPE and T-cells had a major impact on the HFRPE's inhibitory effect. Conclusions. HFRPE cells suppressed the activation of human T-cells by induction of T-cell apoptosis through a process that involves the secretion of soluble factors. The HFRPE mediated T-cell suppression was dependent on the ratio between HFRPE and T-cells. This undefined pathway of T-cell apoptosis may play a role in the maintenance of immune privilege in the subretinal space and may reduce the severity of the immune response after HFRPE transplantation.

Transplantation of Human Fetal Retinal Pigment Epithelium Rescues Photoreceptor Cells from in the Royal College of Surgeons Rat Retina.

Transplantation of Human Fetal Retinal Pigment Epithelium Rescues Photoreceptor Cells from in the Royal College of Surgeons Rat Retina.

Transplantation of Human Fetal Retinal Pigment Epithelium Rescues Photoreceptor Cells from in the Royal College of Surgeons Rat Retina

Transplantation of Human Fetal Retinal Pigment Epithelium Rescues Photoreceptor Cells from in the Royal College of Surgeons Rat Retina

Retinal pigment epithelium cells cultured on synthetic biodegradable polymers.

Alterations in the normal structure or functions of retinal pigment epithelium (RPE) can result in a number of ocular diseases. Implantation of RPE cells cultured on thin, biodegradable polymer films may provide a means of transplanting an organized sheet of RPE cells with distinct apical/basal characteristics for the restoration of normal RPE function. We have investigated the interactions of human RPE cells with different biodegradable polymer films to assess their suitability as substrates for RPE culture. Four biodegradable polymers were used: low molecular weight (MW) 50:50 poly(DL-lactic-co-glycolic acid) (PLGA); high MW 50:50 PLGA; 75:25 PLGA; and poly(L-lactic acid) (PLLA). Polymer film substrates were manufactured using a solvent casting technique. Human fetal RPE cells (10-16 weeks gestational) were plated on the polymer substrates and the cultures assessed with respect to cell attachment and proliferation. Histological and immunohistochemical studies were performed on the cells after 8 days in culture. RPE cells attached to all the polymers studied after 8 h in culture. After 8 h, 80.2 +/- 9.5% and 82.3 +/- 7.9% of the plated cells were attached to substrates of high MW 50:50 PLGA and 75:25 PLGA, respectively. The cells proliferated on all substrates, and there was about a threefold increase in cell number over the 8-day culture period on all the polymers studied. Immunohistochemistry after 8 days in culture demonstrated RPE cells labeled with a distinct reaction product for cytokeratin in the cell cytoplasm. All the polymers studied were suitable for RPE culture; however, high MW 50:50 PLGA and 75:25 PLGA proved to be the best in terms of manufacturing properties, cell attachment, and proliferation. These polymers can provide a suitable substrate for RPE cell culture and hold promise for the subretinal implantation of organized sheets of RPE cells.

Transplantation of Fetal Retinal Pigment Epithelium in Age-Related Macular Degeneration with Subfoveal Neovascularization

Background: Age-related macular degeneration (ARMD) is caused by abnormal retinal pigment epithelium (RPE) and may be complicated by choroidal neovascularization. The object of treatment would be to replace the diseased RPE with normal human RPE. • Method: Five patients with ARMD (preoperative visual acuity 0.08–0.2) underwent removal of subretinal fibrovascular membranes using pars plana vitrectomy techniques. Human fetal RPE (15–17 weeks gestational age) was cultured and transplanted as a monolayer patch into the subretinal space. Transplants were followed by funduscopy and fluorescein angiography. Macular function was assessed using scanning laser ophthalmoscopic (SLO) microperimetry. • Results: Three RPE transplants were placed in the fovea; two were placed parafoveally. All transplants have survived for 3 months. They have grown and increased in size covering part of the epithelial defect caused by removal of the fibrovascular membrane. SLO microperimetry indicated that visual function was present in four of the transplants at 1 month but in only two at 3 months after surgery. Function over the transplants, especially those in the fovea, was compromised by cystoidlike macular edema. • Conclusions: Human fetal RPE transplants survive well in the macula for as long as 3 months. They are capable of growing to cover epithelial defects caused by removal of subretinal neovascular membranes. The causes for development of macular edema in transplants directly in the fovea warrant further evaluation.

Pigment Epithelium‐Derived Factor Is a Survival Factor for Cerebellar Granule Cells in Culture

Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.

Influence of zinc on selected cellular functions of cultured human retinal pigment epithelium.

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coon's modified Ham's F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbecco's modified Eagle's medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.