|Molecular Vision 2006;
Received 23 February 2006 | Accepted 17 August 2006 | Published 29 August 2006
In vitro and in vivo characterization of iris pigment epithelial cells cultured on amniotic membranes
Keisuke Mori,2 Shizuko Ichinose,3 Tetsuji Sato,4
Jiying Wang,1 Noriaki Shimada,1 Ariko Kojima,1 Manabu
Mochizuki,1 Ikuo Morita5
1Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, Tokyo, Japan; 2Department of Ophthalmology, Saitama Medical School, Saitama, Japan; 3Instrumental Analysis Research Center, Tokyo Medical and Dental University, Tokyo, Japan; 4Department of Anatomy, School of Dental Medicine, Tsurumi University, Yokohama, Japan; 5Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo, Japan
Correspondence to: Kyoko Ohno-Matsui, MD. Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8519, Japan; Phone: 81-3-5803-5297; FAX: 81-3-3818-7188; email: firstname.lastname@example.org
Purpose: To determine whether human amniotic membranes (AMs) can induce human and rat iris pigment epithelial (IPE) cells grown on them to develop characteristics of RPE cells in situ better than IPE cells grown on plastic plates, and to determine whether subretinal transplantation of IPE cell sheets grown on AMs can protect photoreceptor cells in dystrophic Royal College of Surgeons (RCS) rats.
Methods: IPE cells from humans and Long-Evans rats were cultured on the basement membrane side of dispase-treated AMs. Two weeks after seeding, ultrastructural changes were evaluated by transmission electron microscopy, and the level of expression of several genes present in differentiated retinal pigment epithelial (RPE) cells was determined by real time PCR and western blotting. IPE cell sheets cultured on AMs were transplanted into the subretinal space of 4-week-old RCS rats, and eyes were analyzed histologically 12 weeks after grafting.
Results: IPE cells cultured on AMs showed ultrastructural features like intercellular junctions, similar to RPE cells in situ. IPE cells grown on AMs had a greater upregulation in the expression of genes important for the function of differentiated RPE cells (e.g., pigment epithelium-derived factor [PEDF], RPE65, bestrophin, VEGF, and BDNF) than IPE cells grown on plastic plates. The number of photoreceptors present in RCS rats after subretinal transplantation of IPE cell sheets grown on AMs was significantly higher than that of sham injected rats and rats receiving transplantation of AMs without IPE cells.
Conclusions: The more advanced degree of differentiation of IPE cells grown on AMs indicates that AMs are a better substrate to culture IPE cells than plastic plates. This was supported by the greater protection of photoreceptors of RCS rats when IPE cell sheets cultured on AMs were transplanted in the subretinal space.
Subretinal transplantation of suspensions of retinal pigment epithelial (RPE) cells has been performed for the treatment of both wet and dry aged-related macular degeneration (AMD). However, the transplantation of suspensions of allogenic RPE cells has been shown to have no visual benefit to patients with AMD because of several factors including graft rejection [1,2]. Because of the rejection, autologous iris pigment epithelial (IPE) cells have been tried to replace the lost or damaged RPE cells in the macular area [3,4]. However, transplantation of suspensions of autologous IPE cells has also not resulted in a prolonged improvement of vision in AMD patients [4,5]. Recently, Binder et al. [6,7] transplanted suspensions of autologous RPE cells into eyes with wet type AMD after removal of choroidal neovascular (CNV) membranes. They reported that these eyes had significantly better reading acuity than controls with CNV removal only. However, obtaining sufficient numbers of RPE cells was sometimes difficult, and in some patients, the aspirated RPE cells were not transplanted because of insufficient numbers or hemorrhage.
In most of these studies, suspensions of isolated cells were injected into the subretinal space. A major problem of injecting suspensions of RPE or IPE cells is that the transplanted cells fail to regain a fully differentiated phenotype. For example, histological examination of the subretinally transplanted IPE cells in rabbits showed that they gradually lose much of their pigment, form multilayered clumps, and survive for only a short time .
The basement membranes of epithelial cells are known to promote the differentiation and survival of epithelial cells [9,10], serve as selective filters, and to have both structural and morphogenetic functions. Bruch's membrane is the basement membrane of the RPE cells and the membrane that the injected cells must attach to. Unfortunately, Bruch's membrane is mechanically damaged when the CNV is removed in patients with wet AMD. In addition, aged human Bruch's membranes do not readily support the subsistence and differentiation of transplanted RPE cells .
To counter these problems, investigators have cultured cells on different types of substrates to form sheets of cells prior to implantation. We have demonstrated that human RPE cells cultured on human amniotic membranes (AMs) developed morphological phenotypes of epithelial cells, and show an upregulation of a panel of growth factors, including pigment epithelium-derived factor (PEDF), a cytokine important for maintaining retinal homeostasis . Stanzel et al.  also demonstrated that RPE adopted an epithelial phenotype with more organized pigmentation, strong expression of ZO-1, and RPE 65 after seeding on AM. AMs are made up of thick basement membranes with an avascular stromal matrix and have been used as a substrate for the transplantation of cultivated corneal epithelial cells .
Because basement membranes of epithelial cells are known to promote the differentiation and survival of epithelial cells [9,10], we have hypothesized that IPE cells grown on human AMs will induce the IPE cells to greater differentiation and stronger upregulation of growth factors than cells grown on plastic plates. To test this hypothesis, we have grown human and rat IPE cells on AMs and on plastic plates and have compared their ability to induce morphological differentiation and upregulation of growth factors from the IPE cells. We also investigated whether subretinal transplantation of IPE cell sheets cultivated on AMs can protect the photoreceptors of Royal College of Surgeon (RCS) rats from degeneration better than sham injected animals.
Human IPE cells were purchased from ScienCell (San Diego, CA). Rat IPE cells were prepared from the eyes of 10 to 12 week-old Long-Evans rats obtained from CLEA Japan (Tokyo, Japan), and the cells were isolated as described . The rat and human IPE cells were maintained in a growth medium consisting of Ham's F-12 (Invitrogen, Tokyo, Japan) and 20% fetal bovine serum. The medium was changed every 2 days, and cells at passage 2 and 3 were used in all experiments. All animal experiments were conducted in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Research Committee, Tokyo Medical and Dental University.
Immunohistochemical detection of cytokeratin
Immunohistochemistry was used to detect cytokeratin, a marker for epithelial cells. For this, rat IPE cells were preincubated in 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 20 min at room temperature (RT). They were then incubated with anti-pan cytokeratins (1:100, Sigma, St. Louis, MO) overnight at 4 °C and then exposed to fluorescein-labeled second antibody in Tween-PBS and 2% FBS for 30 min at room temperature (RT).
Cultures of IPE on AM
Preserved human AMs (AmnioGraft) were purchased from Bio-Tissue (Miami, FL), and the AM was preserved according to the method described by Lee and Tseng . Briefly, AMs derived from Cesarean section-derived placentas were rinsed in phosphate buffered saline (PBS) containing 100 U/ml penicillin and 0.2 mg/ml streptomycin and stored in 50% DMEM and 50% glycerol at -80 °C for up to 3 months. After thawing to room temperature (RT), the AM was treated with 1.2 U/ml sterile dispase-I solution (Godo-Shusei, Tokyo, Japan) for 30 min and then gently scrubbed with an epithelial cell scraper (Costar, Corning, NY), to remove the amniotic epithelium without damaging the underlying basement membrane (denuded AM). The AM was sutured onto a culture insert (Milicell-CM, Millipore, Bedford, MA) with a non-absorbable suture with the basement membrane facing up and placed in a 6 well tissue culture plastic plate as described .
IPE cells were seeded at a density of 1.0x105 cells/AM insert. The cultures were incubated at 37 °C in 5% CO2 and 95% air, and the medium was changed every 2 days. In some experiments, the IPE cells were cultured on uncoated plastic dishes in DMEM with 10% FBS and used as controls. Each condition was prepared in triplicate, and experiments were performed at least three times.
Transmission electron microscopy
Transmission electron microscopy (TEM) was used to determine whether the IPE cells grown on denuded AMs and on plastic dishes differentiated morphologically. Cultures were fixed in 2.5% glutaraldehyde in 0.1 M PBS for 2 h, washed overnight at 4 °C in the same buffer, and post-fixed with 1% osmium tetroxide in 0.1 M PBS for 2 h. The pellets were dehydrated through a graded ethanol series, and embedded in Epon 812. Ultrathin (90 nm) sections were collected on copper grids and double stained with uranyl acetate and lead citrate and examined with a H-7100 TEM (Hitachi Ltd., Hitachinaka, Japan).
cDNA synthesis and SYBR green RT-PCR analysis
Fourteen days after seeding, total RNA was extracted from human IPE cells using TRIzol reagent. Complementary DNAs were synthesized from the total RNA of cultured human IPE cells using a cDNA synthesis kit (You-Prime First-Strand Beads, Amersham Pharmacia, NJ). The SYBR green RT-PCR method (Quantitect SYBR Green PCR KitTM Qiagen Inc., Valencia, CA) was used to determine the expression levels of mRNAs in the cultured human IPE cells using specific primers (Table 1) with a Light CyclerTM instrument (Roche Diagnostics, Basel, Switzerland), as described . The relative cDNA concentrations were determined by a standard curve using sequential dilutions of corresponding PCR fragments. The relative quantity was quantified as the ratio of the mRNA expression of the targeted gene to that of GAPDH.
Western blot analysis of PEDF
Human and rat IPE cells were allowed to condition in serum-free media for 48 h before harvesting to determine the protein concentration. The final protein concentrations were determined using the BCA assay (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. Equal amounts of secreted protein (8 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted onto nylon membranes. Nylon membranes containing transblotted proteins were pretreated with 1.0% non-fat dried milk in 50 mM Tris buffer (pH 8.0), followed by incubation overnight with a monoclonal antibody against human PEDF (dilution 1:4000, Transgenic Co., Kumamoto, Japan). The PEDF immunoreactivity was detected by exposing X-ray film to blots incubated with ECL reagent.
Earlier experiments from this laboratory have shown that human RPE cells will upregulate the expression of PEDF when they are grown on AMs. Thus as a control, experiments were also performed on the supernatants from primary cultures of human RPE cells (generously donated by Peter A. Campochiaro, MD; Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD) at passage 2 to 3. The results were compared with the results of human IPE cells.
Preparation of rat IPE cell sheets on AM for transplantation
Gelatin was used as a matrix to grow rat IPE cells on AMs as has been described for the transplantation of RPE cells sheets by Del Priore et al. . Briefly, porcine skin gelatin powder with a rigidity of 300 blooms (Sigma-Aldrich, St. Louis, MO) was sterilized and dissolved in minimum essential medium (MEM; Invitrogen-Gibco, Grand Island, NY). Sucrose (Sigma-Aldrich) was added to make a 300 mM gelatin solution which maintained the gelatin sheets in a solid phase at temperatures below 37 °C, but the sheets melted within minutes at 37 °C.
Once the gelatin dissolved, the solution was poured into 35 mm tissue culture dishes (Falcon 3001; BD Biosciences, Lincoln Pak, NJ) and allowed to cool and solidify for 15 min at room temperature. Gelatin blocks were cut into 15x30 mm triangular pieces and mounted on a vibratome (Microslicer DSK-3000; Dosaka EM, Kyoto, Japan) with the basal side facing a steel blade. Smooth, 100 μm gelatin sheets were cut from the blocks. The IPE cell sheets cultivated on AMs were placed on a slice of gelatin with the apical IPE surface facing upward. Then, another gelatin film was placed on the IPE cell surface to cover the cell sheet completely. The gelatin film containing the IPE cells attached to the AM was then incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C for 5 min to allow the gelatin to melt and encase the IPE sheet. The specimen was kept at 4 °C for 5 min to solidify the liquid gelatin and then used for transplantation. The viability of the IPE cells after these procedures was more than 90%, as assessed by calcein/AM staining (data not shown).
In some experiments, IPE cells on AMs were labeled with CM-Dil (chloromethylbenz-amino derivatives of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR) before transplantation. For this, the IPE cells were incubated in 5 μg/ml CM-Dil solution for 20 min at 37 °C, and the labeled IPE cells were washed three times with PBS and used for transplantation.
Pink-eyed, dystrophic Royal College of Surgeons (RCS) rats were obtained from the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Japan. Congenic nondystrophic rats were obtained from CLEA Japan (Tokyo, Japan). All transplantations were made into the right eye when the rats were 4 weeks old. The left eye served as untreated controls.
For the injections, animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (20 mg/kg), and the right pupil was dilated using tropicamide (1% Mydrin P, Santen, Tokyo, Japan). The transplantation was performed under an operating microscope (OM-5, TAKAGI, Nagano, Japan). A beveled glass needle (World Precision Instruments, Sarasota, FL) was introduced into the subretinal space transsclerally under direct vision.
The IPE transplant (1x3 mm) was preloaded into the broad end of a beveled glass needle (World Precision Instruments, Sarasota, FL) that was attached to a one ml syringe. To enter the subretinal space, a small incision (about 1 mm) was cut just behind the pars plana of the host eye. The transplant was injected into the subretinal space, and after approximately 10 min, the transplant spontaneously unfolded. In some animals, AM without IPE cells was transplanted as controls.
Sham-treated RCS rats received 1 μl of PBS injected transsclerally into the dorsotemporal subretinal space of the right eye of anesthetized 4-week-old rats by means of a fine glass capillary (inner diameter, 75-150 μm) attached by tubing to a 10 μl syringe (Hamilton, Reno, NV). A total of 32 rats received IPE cell sheet on AM, 12 received AM without IPE cells, and 15 had a sham injection of saline. Transplantation into the subretinal space was confirmed by indirect ophthalmoscopy with a +30 D lens, and those that had successful transplantation were selected for histologic analysis and behavioral tests.
Tissue fixation and processing for histological analysis
Eyes were enucleated 12 weeks after transplantation at age 16 weeks and fixed in 4% paraformaldehyde. The eyes were embedded in OCT compound, and 6 μm sections were cut with a cryostat (Leica CM3050-Cryostat, Wetzlar, Germany), stained, and processed for light microscopy. The maximum thickness of the outer nuclear layer (ONL; n=6 animals for each group) was measured by a single masked observer, and the differences were analyzed with the Mann-Whitney test.
Immunostaining of rhodopsin
The above sections were also used for immunohistochemical analysis. Sections from the periodate-lysine-paraformaldehyde-fixed samples were treated with 0.3% H2O2 and 10% normal horse serum to block endogenous peroxidase and nonspecific binding, respectively. The sections were then treated with mouse monoclonal antithodopsin antibody (1:500 dilution; Sigma-Aldrich, St. Louis, MO) at room temperature for 90 min. After reacting with goat antibodies against mouse IgG conjugated to peroxidase labeled-dextran polymer, the color was developed with aminoethyl carbazole (AEC; Zymed Laboratories, San Francisco, CA) in 50 mM Tris-HCl, containing 0.006% H2O2. Counterstaining was performed with hematoxylin. As a negative control, primary antibodies were replaced with nonimmune mouse IgG (Dako).
Characterization of IPE cells cultivated on AMs
Phase-contrast photographs of isolated rat IPE cells and cultured IPE cells at passage two are shown in Figure 1A,B, respectively. All of the isolated cells are pigmented, and immunohistochemical studies showed that the IPE cells were labeled with antibodies against cytokeratins indicating that they were epithelial origin (Figure 1C).
Morphology of IPE cells grown on AMs
TEM showed that human and rat IPE cells cultured on AM appeared structurally very similar to RPE in situ (e.g., organized in a monolayer with intercellular junctional complexes; Figure 2B,C, Figure 3B,C). In contrast, IPE cells cultured on plastic dishes were not only multilayered but also did not have intercellular junctional specialization of high-electron density (Figure 2A, Figure 3A). Microvilli were sparsely observed on the apical membrane of human IPE cells cultured on AM (Figure 2B).
Reverse transcription PCR
Three discriminatory molecules: RPE65, an RPE-specific molecule that plays an important role in the RPE-photoreceptor vitamin A cycle; cellular retinaldehyde-binding protein (CRALBP) which is involved in the regeneration of visual pigment; and bestrophin, a marker of late RPE differentiation  were investigated in cultured human IPE cells. The expression level of bestrophin was higher in IPE cells cultured on AM than cells cultured on plastic dishes (Table 2). The expression of RPE65 was slightly increased in IPE cells cultured on AM, while the level of mRNA expression of CRALBP was not significantly different between IPE cells grown on AMs and plastic dishes.
The expression of growth and trophic factors which play a role in both RPE and photoreceptor cell function and survival were investigated by RT-PCR. The expression of the mRNA of PEDF was more strongly elevated in IPE cells cultured on AMs than on plastic dishes (Table 2), while there was no significant difference in the mRNA expression of bFGF. The expression levels of VEGF and BDNF were slightly higher in IPE cells cultured on AMs than cultured on plastic dishes.
Tyrosinase and TRP-2 are enzymes involved in melanin synthesis and are expressed in melanocytes of neural crest origin and RPE cells [19,20]. There was no significant difference in the expression of the mRNA of TRP-2, however, that of tyrosinase was slightly upregulated when IPE cells were cultured on AMs.
Western blot of PEDF
Western blot analysis using a monoclonal antibody against purified PEDF protein (Transgenic, Inc., Kumamoto, Japan) was performed. The PEDF antibody recognizes a 50 kDa protein species, and the level of the PEDF protein was almost undetectable in the conditioned medium from IPE cells cultured on plastic dishes, while that from IPE cells cultivated on AMs exhibited a marked increase in PEDF protein production (Figure 4). In our control studies, human RPE cells were found to produce significantly higher levels of PEDF when grown on AMs than on plastic plates, as we have already reported (Figure 4A). PEDF production of human IPE cells on AMs was still lower than that of human RPE cells both on plastic dishes and on AMs.
Photoreceptor rescue by subretinal transplantation of IPE cell sheets grown on AMs in RCS rats
RCS rats show a progressive loss of photoreceptors, which is most marked during the first three months after birth . This retinal degeneration is primarily due to the failure of the RPE cells to phagocytose shed photoreceptor outer segments . Previous studies have demonstrated that subretinal transplantation of fetal RPE cells into the dystrophic RCS rat at an early age resulted in structural and functional preservation of photoreceptors [23-25].
We used this animal model to explore the ability of IPE cells cultured on AM to rescue photoreceptor degeneration in RCS rats. The IPE cell sheets grown on AMs were successfully injected subretinally in 23 (71.9%) of 32 eyes without significant complications. The other nine eyes had extensive vitreous hemorrhage (five eyes), retinal detachment (two eyes), cataract formation (one eye), and endophthalmitis (one eye), and were excluded from further analysis. The AMs without IPE cells were successfully injected subretinally in 10 of 12 rats. The other two eyes had cataract formation, and were excluded from analysis. In the sham surgery eyes, none showed significant complications. When the animals were 16 weeks old (12 weeks after transplantation), the eyes were removed and were processed for histological analysis.
There was no histological evidence of an inflammatory reaction or infiltrating immune cells in any of the experimental or control eyes. Light microscopic observation revealed that the transplanted tissue was located subretinally (Figure 5D), and was easily recognized by the melanin pigments (Long-Evans donor) from the host's unpigmented RPE cells. Prelabeling of the IPEs with CM-DiI confirmed that these heavily pigmented cells in the host subretinal space were derived from the donor cells (Figure 5E). The subretinally transplanted IPE cells appeared to be round in shape (Figure 5D).
Histological analysis of nondystrophic rat retinae showed an organized row of nuclei in the outer nuclear layer (ONL) of about 14 nuclei thick (Figure 5A), but in the unoperated eye of dystrophic rats, the ONL was reduced to an occasional cell lying at the outer border of the inner nuclear layer (Figure 5B). Animals that had sham surgery had findings similar to those of unoperated rats, and almost no photoreceptors remained (data not shown). Animals that had transplantation of AMs without IPE cells showed only about 1-2 nuclei thick in the ONL (Figure 5C).
In contrast, more photoreceptor nuclei were present in the group of animals that received IPE cell sheets grown on AMs (Figure 5D). Photoreceptor preservation was also observed in the areas that were not immediately overlying transplanted IPE cells (data not shown). Retinae that had received transplants showed larger areas of photoreceptor survival with the ONL as much as eight nuclei thick in the area of the injection (Figure 5F). There was a significant lower number of nuclei in the ONL of rats that had no treatment or sham surgery than animals that underwent transplantation of IPE cell sheet on AMs (p<0.05). There was also a significantly lower number of nuclei in the ONL of rats that received transplantation of AMs without IPE cells than those that received transplantation of IPE cell sheets grown on AMs (p<0.05). There was no significant difference in the number of nuclei in the ONL in untreated rats from rats that had sham surgery.
Immunohistochemical analysis showed that the preserved photoreceptors expressed rhodopsin, a visual pigment used by the rod photoreceptor cells to perform phototransduction (Figure 6).
IPE cells have recently been used for autologous cell transplantation for retinal diseases, however the transplantation did not result in a prolonged improvement of vision in AMD patients [4,5,26]. One of the factors for this failure was probably because the transplanted IPE cells did not fully differentiate into cells that had morphological and physiological properties of RPE cells in situ. To determine whether the differentiation was important, we hypothesized that IPE cells grown on human AMs will induce the IPE cells to differentiate better because we had demonstrated earlier that human RPE cells cultured on AMs developed morphological phenotypes of epithelial cells, and show an upregulation of a panel of growth factors . Our results supported our hypothesis, as IPE cells cultured on AMs exhibited morphological features and gene expression profiles more comparable to differentiated epithelial cells than did IPE cells grown on uncoated plastic dishes. Ultrastructurally, IPE cells cultured on AMs were organized into a tight monolayer with distinct intercellular junctions, while IPE cells cultured on uncoated plastic dishs were elongated and organized in multilayered clumps. In addition to the morphological features, IPE cells cultured on AMs showed a marked upregulation of PEDF expression compared to cells grown on plastic dishes.
Semkova et al.  reported that PEDF was not present in the supernatants of cultured IPE cells using ELISA (detection limit 1.56 ng/ml), and they suggested that the lack of PEDF expression might be the main cause of the failure of prolonged improvement of vision in AMD patients who received subretinal transplantation of autologous IPE cells. The finding that subretinal transplantation of autologous IPE cells genetically-modified to express PEDF increased the survival of photoreceptor cells in RCS rats supported their hypothesis . In our study, the level of PEDF was almost undetectable in human and rat IPE cells cultured on plastic dishes, however, the level of PEDF protein was significantly higher in IPE cells cultured on AMs. We have already shown that PEDF is not released from AMs after removal of the amniotic epithelial cells .
As far as we know, this is the first study demonstrating the production of PEDF by IPE cells. Our data suggest that the expression of PEDF by IPE cells is regulated by the phenotypic changes as we previously demonstrated for RPE cells [12,28]. PEDF is a critically important factor of RPE cells to maintain retinal homeostasis, because it has strong antiangiogenic  and neurotrophic [30,31] activities in the eye. Thus, the marked increase in PEDF expression in IPE cells grown on AMs should aid in the success of transplantation of IPE cell sheets grown on AM, as suggested by Semkova et al. .
To determine whether differentiation of the IPE cells had functional consequences, we transplanted IPE cell sheets cultivated on AMs and on plastic plates into the subretinal space of RCS rats. Our histological analysis showed that there were significantly more nuclei in the ONL in eyes that had received a transplantation of IPE cells grown on AMs than eyes from sham injected animals and animals receiving transplantation of AMs without IPE cells. Also, immunohistochemical analysis showed that the preserved photoreceptors expressed rhodopsin. These results indicate that the IPE cells cultured on AM may develop a capability to promote the survival of photoreceptor cells in an animal model of RPE dysfunction. Growth factors released from transplanted IPE cells may play a role in photoreceptor preservation.
Although it is not clear which components of AM are responsible for controlling the phenotypic alterations of IPE, growth factors contained in AM or extracellular matrix components of AM might have a role, as discussed previously .
However, there still are other differences between IPE cells and RPE cells in the phenotypic changes induced by AM. Gene expression analysis using real time PCR demonstrated that the levels of mRNA of RPE65, VEGF, and BDNF were only slightly increased, and those of CRALBP was not different for IPE cells grown on AMs and on plastic plates. In contrast, the expression levels of all of these genes were markedly upregulated in differentiated RPE cells cultured on AM compared to cells grown on plastic dishes . RPE65 and CRALBP are well-known markers for differentiated RPE cells [32,33], and VEGF and BDNF are important trophic factors for maintaining retinal homeostasis [34,35]. Only PEDF was dramatically upregulated in IPE cells cultured on AMs, however, western blot analysis using supernatants from both human RPE cells and IPE cells demonstrated that the PEDF protein levels in IPE cells cultured on AMs were still lower than even that from nondifferentiated RPE cells on uncoated plastic plates (Figure 4A). These results suggest that IPE cells might not be able to fully reproduce the features of differentiated RPE cells even when they are manipulated to induce phenotypic changes by the AMs. Although IPE cells have the same embryogenic origin as RPE cells, these data suggest that there might be some biochemical differences between IPE and RPE cells. Also, in vivo transplanted IPE cells appeared round in shape in the subretinal space. The round morphology of transplanted IPE cells was similar to that observed in the study by Scraermeyer  who performed subretinal injection of IPE cell suspension to RCS rats. This suggests that merely culturing of IPE cells on AMs might not be sufficient to induce cell adhesion and cell survival.
The average thickness of the AMs as used in our study (AmnioGraft) was reported to be 32.13 μm . This relatively thick membrane could reduce or prevent blood products and oxygen from diffusing to the transplanted cells from the underlying choroid. In addition, AMs are very sticky and difficult to handle during the implantation. A better substrate to solve these problems and to stimulate the formation of a monolayer of fully functioning cells with differentiated properties is the goal of our future studies.
In conclusion, our results suggest that AMs are a good substrate to induce differentiation of IPE cells for subretinal transplantation. AMs induced morphological changes as well as marked upregulation of PEDF expression. In addition, subretinal transplantation of IPE cell sheets maintained on AMs had a protective effect on the photoreceptor cells of RCS rats. However, further studies are necessary before IPE cells are transplanted to replace damaged RPE cells.
The authors thank Drs. Hackett and Campochiaro for human RPE cells (primary culture, passage 1). We also thank Yoshio Miki for gene analysis, Masatoshi Haruta for advice, Tomoko Yoshida for excellent technical support, and Dr. Duco Hamasaki for editing the manuscript. This work was partly supported by research grant 16390495 and academy frontier project 2003-2007 from the Japan Society for the Promotion of Science.
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