Iris Pigment Epithelial Cells Research Articles

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.

Establishment of Effective Methods for Transducing Genes into Iris Pigment Epithelial Cells by Using Adeno-associated Virus Type 2

To establish an efficient method of transferring the human brain-derived neurotrophic-factor (hBDNF) gene into human iris pigment epithelial (hIPE) cells by using recombinant adeno-associated virus type 2 (rAAV2). Cultured hIPE cells were treated with either hydroxyurea-sodium butyrate (HUSB; DNA synthesis inhibitor), or tyrphostin-1 (Tyr; epidermal growth factor receptor [EGFR] tyrosine kinase inhibitor), or a combination of HUSB and Tyr (HUSB-Tyr). After each treatment, cells were exposed to rAAV2 (rAAV-LacZ or rAAV-hBDNF). The levels of BDNF were measured by ELISA and also determined by Western blot analysis. Southern blot analysis was performed on each type of treated cell. The neuroprotective effect of BDNF on the retinal ganglion cells (RGCs) was quantitatively assessed by culturing rAAV-hBDNF-hIPE with RGCs. The infection of hIPE cells was significantly lower than ARPE and HT1080 cells, which are highly permissive cells for rAAV2. The treatment of HUSB-Tyr enhanced the transgene expression more than that after treatment with one of these agents in rAAV-hIPE cells. Southern hybridization revealed that the amount of replicative form monomer (RFm) was less in Tyr than in HUSB or HUSB-Tyr treatment and there was no difference in conversion of virus genome to double stranded form after HUSB and HUSB-Tyr treatment. However, adding Tyr treatment stimulated the JNK1/2 and p38 pathways and modified the target transgene expression. BDNF had a significantly greater rescue effect of RGCs with the HUSB-Tyr-treated rAAV-hBDNF-hIPE cells (P < 0.01) than that with the HUSB-treated rAAV-hBDNF-hIPE cells (P > 0.05) compared with noninfected hIPE cells. The combined treatment of HUSB-Try is an effective method of increasing transgene expression with the AAV-mediated gene transfer. The role of HUSB and Tyr in the increase of gene expression may be different and related to the conversion of virus into the host genome and the enhancement of the transcription, respectively.

Iris Pigment Epithelium Attachment to Aged Submacular Human Bruch’s Membrane

To determine whether iris pigment epithelium (IPE) cells can attach to aged submacular human Bruch's membrane and to assess whether IPE cells express the integrin subunits that may be necessary to bind to the known extracellular matrix ligands present in Bruch's membrane. IPE cells were seeded onto the RPE basement membrane (RPEbm) or inner collagenous layer (ICL) of aged submacular Bruch's membrane as microaggregates or were expanded in culture until enough cells could be obtained for seeding. Cell morphology and the percentage of cell coverage were determined 1 or 7 days after seeding. Messenger RNA was extracted from cultured and uncultured IPE cells and analyzed by RT-PCR. The expression of integrin subunits alpha1 to alpha6 and beta1 mRNA was examined. Coverage by uncultured IPE was low on both surfaces at day-1 (RPEbm, 7.9% +/- 4.8%; ICL, 5.0% +/- 2.5%) with few intact cells present. Culturing IPE improved attachment with similar coverage on both surfaces and no significant difference between day-1 (RPEbm, 89.9% +/- 9.1%; ICL, 63.4% +/- 26.5%) and day-7 (RPEbm, 97.8% +/- 2.3%; ICL, 94.7% +/- 6.6%). By day-7, cell morphology and coverage on both surfaces was variable, ranging from few intact cells to a high degree of coverage by flattened cells. All integrin subunits studied were expressed in cultured cells, whereas alpha2, alpha3, and alpha4 showed less or no expression in uncultured cells. Upregulation of integrin mRNA expression may be one explanation for the difference in coverage by cultured versus uncultured IPE cells. The presence of dead, dying, or flattened cells at day 7 indicates that IPE may not survive or differentiate on aged submacular Bruch's membrane.

Photoreceptor protection by iris pigment epithelial transplantation transduced with AAV-mediated brain-derived neurotrophic factor gene.

To determine whether subretinal transplantation of iris pigment epithelial (IPE) cells transduced with the adeno-associated virus (AAV2)-mediated brain-derived neurotrophic factor (BDNF) gene can protect photoreceptors against phototoxicity. The BDNF gene was inserted into AAV2 (AAV2-BDNF), and the recombinant AAV2 was transduced into rat IPE (AAV2-BDNF-IPE) cells at various multiplicities of infection (MOI). The concentrations of AAV capsids and BDNF were determined by enzyme-linked immunosorbent assay (ELISA). The AAV2-BDNF-IPE cells were transplanted into the subretinal space of rats, and the rats were placed under constant light on days 1 and 90 after the transplantation. The thickness of the outer nuclear layer was measured in histologic sections and compared to that of control sections. The expression of beta-galactosidase (LacZ) in the subretinal space was confirmed by LacZ staining after AAV2-LacZ-IPE transplantation. BDNF gene expression after transplantation was confirmed by real-time polymerase chain reaction (PCR). Transduction efficiency increased with successive days in culture and increased with higher MOI in vitro. The expression of the BDNF gene in the subretinal space was higher in AAV-BDNF-IPE than with AAV2-LacZ-IPE or with IPE-only transplantation. LacZ expression was observed in the subretinal space 7 and 90 days after transplantation. A statistically significant photoreceptor protection was observed on days 1 and 90 in eyes receiving the AAV2-BDNF-IPE transplant, in both the superior transplant site and the inferior hemispheres which did not receive the transplant. Transplantation of AAV2-BDNF-IPE cells may be an alternative method of delivering neurotrophic factors to the lesion.

Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.

Latanoprost-induced pigmentation in human iridial melanocytes is fibroblast dependent

The prostaglandin F2α derivative, latanoprost (LT), used in glaucoma treatment, can induce pigmentation in irises of patients with hazel or heterochromatic eye colour. The mechanism by which LT induces pigmentation in the iris is not yet established, although it does not appear to induce proliferation of iridial melanocytes. The purpose of this study was to develop an in vitro model in which to investigate this mechanism. The pigmentary responses to LT and prostaglandin F 2α (PGF 2α) were examined in human iridial melanocytes alone or in co-culture with epithelial cells (non-ocular human epidermal keratinocytes and iris pigment epithelial cells) or mesenchymal cells (non-ocular dermal fibroblasts or iridial fibroblasts). Melanogenesis was assessed after 4 days culture with prostanoids, using dopa oxidase activity. Prostaglandin FP expression on human iridial fibroblasts and melanocytes was investigated using an immunofluorescent technique employing antibody to PGF 2α receptor and RT-PCR. Iridial melanocytes did not show a convincing increase in dopa oxidase when cultured alone but in the presence of fibroblasts (ocular or non-ocular) there was a significant increase (25–30%) in dopa oxidase activity in response to 10 −7–10 −5 m LT and PGF 2α. Co-culture of melanocytes with epithelial cells, while leading to increased dopa oxidase activity, did not lead to any melanogenic response to LT or PGF 2α. FP receptor expression was detected on fibroblasts but not iridial melanocytes by immunocytochemistry and RT-PCR. The melanocyte/fibroblast co-culture model developed in this study also showed that LT and PGF 2α increased dopa oxidase activity in melanocytes from donors with brown but not blue eyes. These results suggest that LT may be inducing pigmentation in the human iris indirectly through the FP receptor on adjacent fibroblasts.

Adeno-associated virus mediated delivery and expression of GDNF cDNA in cultured bovine iris pigment epithelial cells

To obtain transgenic bovine iris pigment epithelial cells (IPECs) by adeno-associated virus (AAV) mediated delivery of cDNA of glial cell-line derived neurotrophic factor (GDNF). AAV-GDNF was titrated by slot blotting. Cultured bovine passage two IPECs were transfected using AAV-GDNF at dosage of MOI (multiplicity of infection) = 50, then were cultured in DMEM medium complemented with 3% FBS for 4 weeks with no change of medium. The expression of GDNF in culture medium was examined using ELISA test. By using the same methods, cultured passage two IPECs were transfected with AAV-GFP (green fluorescent protein) at dosage of MOI = 50, and then were cultured in DMEM medium complemented with 20% FBS. The expression of GFP in IPECs was examined using fluorescence microscope every 2 days after transfection. (1) GFP expression in IPECs could not be detected until 4 days after transfection. The positive GFP expression in IPECs reached fastigium in day 8 or 10. (2) According to the results of ELISA test, concentration of GDNF in the culture medium was (17.14 +/- 1.10) micro g/L. AAV-GDNF can effectively transfect cultured IPECs, and the transgenic cells show a high expression of GDNF.

A newt's eye view of lens regeneration

In this paper we describe the basic process of lens regeneration in adult newt and we pinpoint several issues in order to obtain a comprehensive understanding of this ability, which is restricted to only a few salamanders. The process is characterized by dynamic changes in the organization of the extracellular matrix in the eye, re-entering of the cell cycle and dedifferentiation of the dorsal iris pigment epithelial cells. The ability of the dorsal iris to contribute to lens regeneration is discussed in light of iris-specific gene expression as well as in relation to factors present in the eye.

Iris pigment epithelium expressing CD86 (B7-2) directly suppresses T cell activation in vitro via binding to cytotoxic T lymphocyte-associated antigen 4.

A monolayer of pigment epithelium (PE) lines the iris PE (IPE), ciliary body PE, and retina PE of the inner eye, an immune-privileged site. These neural crest-derived epithelial cells participate in ocular immune privilege through poorly defined molecular mechanisms. Murine PE cells cultured from different ocular tissues suppress T cell activation by differing mechanisms. In particular, IPE cells suppress primarily via direct cell to cell contact. By examining surface expression of numerous candidate molecules (tumor necrosis factor receptor [TNFR]1, TNFR2, CD36, CD40, CD47, CD80, CD86, PD-L1, CD95 ligand, and type I interferon receptor), we report that IPE cells uniquely express on their surface the costimulatory molecule CD86. When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation. IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice. We conclude that iris pigment epithelial cells constitutively express cell surface CD86, which enables the cells to contact inhibit T cells via direct interaction with CTLA-4. Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation.

Comparative study of cathepsins D and S in rat IPE and RPE cells

To investigate differences between activities related to phagocytosis in iris pigment epithelial (IPE) and retinal pigment epithelial (RPE) cells, an aspartic protease, cathepsin D (cat D), and a cysteine protease, cathepsin S (cat S), of IPE and RPE were studied. IPE and RPE cells were isolated from Long Evans rat eyes. The origin of the isolated cells was determined by pigmentation and cytokeratin labelling. The mRNA expressions of cat D and cat S in cultured IPE or RPE cells were investigated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Enzyme activities of cat D and cat S in IPE or RPE cells were measured by using specific fluorogenic substrates, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-(Dnp)D-Arg-NH2 and Z-Val-Val-Arg-MCA, respectively. Western blot analysis of both proteins was also performed. The cultured cells, both of IPE and RPE cells were pigmented and showed positive labelling with an anti-cytokeratin monoclonal antibody. The cat D activity in RPE cells was 37 times that in IPE cells. The cat S activity in RPE cells was four times that in IPE cells. On the other hand, mRNA expression levels of cat D in RPE cells were at the same level with IPE cells, cat S mRNA expression in RPE cells were 10 times that in IPE cells. These results were also correlated with the Western blot analysis. In this study, we measured the characteristic expressions of cat D and S in IPE and RPE cells for the first time to compare their lysosomal activities. IPE cells have the lysosomal activities like RPE cells, however, the function of lysosomal activity in IPE cells is beneath RPE's. These results indicated that the ability of ROS digestion in IPE cells was not same as RPE cells.

Age-Dependent Iris Abnormalities in Collagen XVIII/Endostatin Deficient Mice with Similarities to Human Pigment Dispersion Syndrome

Collagen XVIII is expressed in ocular basement membranes (BMs) and inactivating mutations cause Knobloch syndrome, with several ocular abnormalities. In this study we investigated ocular structures in collagen XVIII/endostatin (Col18a1(-/-))-deficient mice to elucidate the role of this extracellular matrix component in the eye. Eyes of Col18a1(-/-) and control mice were examined by light and transmission electron microscopy, laser scanning ophthalmoscopy, and fluorescence angiography. Immunohistochemical analysis of neuronal, epithelial, and immune cells in the eye was performed with antibodies against established cell markers. Col18a1(-/-) mice showed a disruption of the posterior iris pigment epithelial (IPE) cell layer with release of melanin granules. The BM of the posterior IPE was attached to the lens and the nonpigmented epithelium of the ciliary body, which was flattened in mutant mice. In aged mutant mice a severe thickening of the stromal iris BM zone was found, and pigmented cells migrated out of the iris and covered the retina along the inner limiting membrane (ILM), sometimes penetrating into the retina. These cells resembled iris clump cells, and immunohistochemistry demonstrated that they were macrophage-like cells. Furthermore, morphologically abnormal retinal vasculature was seen by fluorescence angiography. The abnormalities in the iris and ciliary body of Col18a1(-/-) mice demonstrate an important role of collagen XVIII for the function of ocular BMs. The absence of this collagen alters the properties of BMs and leads to severe defects in the iris, showing striking similarities to human pigment dispersion syndrome. In addition, loss of collagen XVIII creates changes that allow clump cells to migrate out of the iris. These cells have not been well characterized previously. In the current study we showed that they are macrophage-like cells and are able to penetrate the ILM in mutant mice. The disease mechanism of human pigment dispersion syndrome is not well understood, but Col18a1(-/-) mice may serve as a model and demonstrate the potential importance of alterations in extracellular matrix components in this disease.

Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye.: Semkova I, Kreppel F, Welsandt G, Luther T, Kozlowski J, Janicki H, Kochanek S, Schraemeyer U. Proc Nat Acad Sci U S A 2002;99:13090–13095

Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye.: Semkova I, Kreppel F, Welsandt G, Luther T, Kozlowski J, Janicki H, Kochanek S, Schraemeyer U. Proc Nat Acad Sci U S A 2002;99:13090–13095

Expression and functional properties of unique inward rectifier K+ channel Kir7.1 in the porcine iris and retinal pigment epithelium

Purpose. To investigate the membrane functional properties of porcine iris pigment epithelial cells (IPE), and compare the characters of inward rectifier potassium (Kir) channel in the IPE with those in the retinal pigment epithelial cells (RPE). Methods. IPE and RPE were acutely dissociated from porcine eyes. Functional properties of Kir channels were characterized using whole cell patch clamp recording techniques. Expression of Kir7.1 mRNA in both cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results. Whole cell current in the IPE exhibited a mild inward K + rectification, and showed little dependence on [K + ] o. Unusual high (7.04 ± 1.7) Rb + to K + inward conductance ratio indicated that Kir7.1 subunit was expressed in the IPE as the same as RPE cells. Also, Kir7.1 mRNA was detected in both porcine IPE and RPE by RT-PCR. However, functional expression of Kir conductance in IPE cells (21.7 S/F) was much smaller than that in RPE cells (205.6 S/F). Conclusions. The Kir7.1 subunit was predominantly expressed in the acutely dissociated porcine IPE and its functional properties are similar to those in the RPE. However, the current density seems too small to fulfill the task of the Kir function of RPE.

細胞治療とＤＤＳ 網膜組織再生治療へのアプローチ

Retinal degenerative disease such as retinitis pigmentosa and age-related macular degeneration causes visual defects with the progress in their diseases. In case of retinitis pigmentosa a large number of mutations in genes that involved in phototransduction pathway have been identified. However pharmacological treatment is presently unavailable for these diseases. Several strategies such as retinal pigment epithelial cell transplantation, growth factor application, vitamin supplementation or gene therapy are under examination for preventing the photoreceptor cell death. Among these strategies, some growth factors are commonly effective on preventing photoreceptor cell death even the mutated genes cause that photoreceptor cell death is different. Is it possible to prevent photoreceptor cell death and to maintain the visual function by continuously supplying growth factors to photoreceptor cells? Which methodology is better to supply growth factor continuously, gene transfer to retina or transplantation? We describe here the approach to prevent photoreceptor cell death by using gene transfected iris pigment epithelial cell transplantation.

Microcontact printing on human tissue for retinal cell transplantation.

To demonstrate that microcontact printing, a modern materials fabrication technique, can be used to engineer the surface of human tissue and to show that inhibitory molecules can be used to pattern the growth of retinal pigment epithelial cells or iris pigment epithelial cells on human lens capsule for transplantation. Photolithographic techniques were used to fabricate photoresist-coated silicon substrates into molds. Poly(dimethylsiloxane)stamps for microcontact printing were made from these molds. The poly(dimethylsiloxane) stamps were then used to "wet-transfer" growth inhibitory molecules to the surface of prepared human lens capsules that were obtained during cataract surgery. Human retinal pigment epithelial and rabbit iris pigment epithelial cells were grown on a lens capsule substrate in the presence and absence of a patterned array of inhibitory factors. We found that human lens capsule could be microprinted with a precision similar to that obtained on glass or synthetic polymers. Retinal pigment epithelial cells and iris pigment epithelial cells cultured onto an untreated lens capsule showed spreading and formed into fusiform-appearing cells. In contrast, cells cultured on a lens capsule with a hexagonal micropattern of growth inhibitory molecules retained an epithelioid form within the inhibitory hexagons. Inhibitory growth molecules can be micropatterned onto human lens capsule, and these micropatterns can control the organization of retinal pigment epithelial cells or iris pigment epithelial cells cultured onto the lens capsule surface. Microprinting on autologous human tissue may facilitate efforts to effectively organize cell cultures and transplantations for the replacement of vital ocular tissues such as the retinal pigment epithelium in age-related macular degeneration.

Acta Ophthalmologica Award and the KKK Lundsgaard Prize 2002

At the recent XXXVth Nordic Congress of Ophthalmology the new Acta Ophthalmologica Award was given to Professor Niels Ehlers, Århus, Denmark. The new Acta Ophthalmologica Award is given as a recognition for excellence in ophthalmic research to a scientist from the Nordic countries. It was first granted this year, and will be handed out at every Nordic Congress of Ophthalmology in the future. The Acta Ophthalmologica Award involves not only the honour and the right to give the Acta Honorary Lecture, but the recipient of the reward also receives a gold medal and a cheque of 50,000 Danish Crowns. The medal depicts the same two owls, that have always been associated with Acta Ophthalmologica (Fig. 2). The owls come from a small Dutch painting in Statens Museum for Kunst in Copenhagen. The Acta Ophthalmologica Award The Acta Ophthalmologica Board of Directors select the recipient. This Board has two members from each of the Nordic countries, and the academic ophthalmic communities of these countries are also invited to nominate the awardee. Professor Niels Ehlers, has published over 180 papers and is a leading expert on corneal disease and eye banking internationally. He has written extensively onthese subjects, both clinical and basic science papers. Professor Ehlers has also made very important contributions in ophthalmic fields that are totally unrelated to the cornea, e.g. ocular tumours, particularly retinoblastoma, tapetoretinal degeneration and glaucoma. Professor Ehlers, has previously received eight awards, among those the von Helmholtz Medal and Professor Schiøtz Memory Medal. Professor Ehlers served as Chief Editor for Acta Ophthalmologica between 1987 and 2000 and has also been a member of the Editorial Board of six other ophthalmic scientific journals. At the same congress the KKK Lundsgaard prize for 2002 was given for the best article published in Acta Ophthalmologica during 2000 and 2001, a unanimous decision by the Board of Directors after nominations from all Nordic Professors of Ophthalmology. The prize winners were Sven Crafoord, Lijun Geng, Stefan Seregard and Peep Algvere for their article ‘Experimental transplantation of autologous iris pigment epithelial cells to the subretinal space’. Each author received the KKK Lundsgaard Medal in silver.

Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye.

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders.

Photoreceptor survival in transplantation of autologous iris pigment epithelial cells to the subretinal space.

To investigate photoreceptor survival in transplantation of non-cultured iris pigment epithelial (IPE) cells to the subretinal space in a prospective experimental study. Upper iridectomies were carried out in the right eyes of 37 pigmented rabbits. Suspensions of freshly harvested autologous IPE cells (without culturing) were prepared and injected into the subretinal space of the same eye. Follow-up examinations were carried out using ophthalmoscopy and colour fundus photography. The rabbits were killed at 1, 2, 3 and 6 months, respectively, and the eyes examined with light and electron microscopy. On histological examination, the photoreceptor cells were found to be well-preserved in grafted areas at 1-3 months. At 6 months, the photoreceptors generally disclosed a normal nuclear layer and long outer segments when overlying areas with single cells or clusters of transplanted IPE cells. Multilayers of cells in abundance, including native RPE cells and macrophages (stained with RAM 11), particularly under microfolds of the neural retina, were occasionally associated with photoreceptor damage and nuclear drop out from the outer retinal layer. There was no inflammatory response in the choroid and the choriocapillaris remained patent. The experiments show that grafting freshly harvested autologous IPE cells to the subretinal space is feasible and that the photoreceptors generally survive for at least 6 months when overlying the transplanted areas. Multilayers of abundant cells in the subretinal space may induce adverse focal effects on adjacent photoreceptors.

Iris pigment epithelial cells transplanted into the vitreous accumulate at the optic nerve head.

Iris pigment epithelial (IPE) cells have mainly been investigated in the past for their proposed potential to rescue or even replace degenerated retinal pigment epithelial (RPE) cells after subretinal transplantation in patients with age-related macular degeneration (AMD). More recent reports have characterised the IPE cell as a potent source of trophic factors and cytokines. In our study we investigated the spatial distribution of IPE cells that were injected into the vitreous instead of being injected subretinally. IPE cells from Long Evans rats were isolated and injected into the vitreous cavity of Wistar rats without preculturing. Free melanin granules were injected into the vitreous in the same manner. After a period of 2 months, eyes were prepared for histological analysis. Localisation of the injected IPE cells was defined by topographical mapping of the analysed sections. PVR was not observed in any eye. In 8 of 10 injected eyes, IPE cells had accumulated in the prepapillary region. In 2 of 10 eyes, no IPE cells could be detected. The injected melanin granules also accumulated at the optic nerve head, indicating that this is most likely a passive process. In sections of the papillary region containing retinal vessels, the IPE cells seemed to have migrated into the superficial tissue of the optic nerve head. Our results demonstrate a way to access the optic nerve head easily and securely without the danger of damaging its fragile structure. This could have important implications for new therapeutic strategies in ocular neurodegenerative diseases like glaucoma. New prospects in gene therapy will require further characterisation of the potential of the IPE cell to produce neuroprotective trophic factors at the optic nerve head.

Autologous subretinal transplantation of cultivated porcine iris pigment epithelial cells (IPE)

Subretina transplantation of epithelium may be a therapeutic option for surgical treatment of age-related macular degeration (AMD). Various experimental data have demonstrated that homologous transplantation of retinal pigment epithelium (RPE) can prevent photoreceptor deterioration. However, most investigators experienced immunogenic graft rejection when using homologous pigmented cells for grafting. Autologous cells were soon considered as an alternative for subretinal grafting. Particularly iris pigment epithelium (IPE) appeared suitable to replace homologous RPE for it embryogenetic similarity and its simple availability. Recent studies have shown, that IPE is capable of taking over functions of RPE in maintaining retinal metabolism. the purpose of this study was to evaluate if autologous IPE cells would survive when being transplanted subretinally. In addition, immunogenic reponses to the presence of "foreign" iris pigment cells needed to be excluded. Iris tissue was obtained by peripheral iridectomy in the anesthetized pig. Sheets of pigment iris epithelium were separated from the specimens and transferred into tissue culture. After the cells had been grown to confluency, cell suspensions were injected into the subretinal space of the donor animal's fellow eye. After 4 weeks, the grafted eye was enucleated and examined histologically.. The histological exam revealed that the graft cells had survived in the subretinal space. No evidence of immunogenic rejection was observed. Autologous IPE-cells can survive in the host's sub-retinal space without creating inflammatory reactions. Transplanted IPE appears to interact with photoreceptor outer segments.