CULTURE OF RPE CELLS

Abstract

The invention relates to the use of sericin in the culture of retinal pigment epithelium (RPE) cells or RPE precursor cells, wherein sericin is added to the culture medium to promote pigmentation of cultured RPE cells or RPE precursor cells. The invention further relates to the use of said RPE cells or RPE precursor cells for use in therapy.

Claims

1. Use of sericin in the culture of retinal pigment epithelium (RPE) cells or RPE precursor cells, wherein sericin is added to the culture medium to promote pigmentation of cultured RPE cells or RPE precursor cells.

2. The use of claim 1 wherein the culture medium is serum-free.

3. The use of claim 1 or claim 2 wherein the medium is a chemically-defined medium.

4. The use of any one of claims 1 to 3 wherein the medium is or comprises a minimal medium.

5. The use of any one of claims 1 to 4 wherein the medium is or comprises a Minimal Essential Medium.

6. The use of claim 5 wherein the minimal medium is selected from Eagles Minimal Essential Medium, Minimum Essential Medium Eagle with modification (MEM), Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F12, IMDM, Medium 199, Medium 109, RPMI 1640, Ham F10, Ham F12, and McCoy's 5A.

7. The use of claim 6 wherein the minimal medium is or comprises MEM or DMEM with high glucose and pyruvate.

8. The use of any one of claims 1 to 7 wherein the concentration of sericin is 0.01 to 10% (w/v), preferably 0.1 to 5, 0.1 to 4, 0.1 to 3, or 0.1 to 2% (w/v), preferably 1% (w/v).

9. The use of any one of claims 1 to 8 wherein the medium further comprises one or more additives selected from an antibiotic, a pH indicator, a hormone, a growth supplement, taurine, a non-essential amino acid and glutamine.

10. The use of claim 9 wherein the antibiotic is penicillin and/or streptomycin.

11. The use of claim 9 or claim 10 wherein the pH indicator is phenol red.

12. The use of any one of claims 9 to 11 wherein the hormone is hydrocortisone and/or triiodo-thyronine.

13. The use of any one of claims 9 to 12 wherein the growth supplement is N1 medium supplement which comprises transferrin, insulin, putrescine, progesterone, selenium and biotin.

14. The use of any one of claims 9 to 13 wherein the non-essential amino acid is one or more selected from alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, asparagine, and selenocysteine.

15. The use of any one of claims 1 to 14 wherein the medium is MEM comprising N1 growth supplement, taurine, triiodo-thyronine, one or more non-essential amino acids, glutamine, penicillin, streptomycin and hydrocortisone.

16. The use of any one or claims 1 to 14 wherein the medium is DMEM with high glucose and pyruvate comprising penicillin and streptomycin.

17. The use of any one of claims 1 to 16 wherein the RPE cells are cultured for up to 60 days, preferably for up to 30, 21 or 14 days.

18. A method for the culture of RPE cells or RPE precursor cells, said method comprising culturing said cells in a medium comprising sericin, wherein the sericin promotes pigmentation of the cultured RPE cells or RPE precursor cells.

19. The method of claim 18, wherein the medium is as defined in any one of claims 2 to 16.

20. An RPE cell or population of cells produced by the use or method of any one of claims 1 to 19.

21. The RPE cell or cell population of claim 20 for use in therapy, preferably for use in treating or preventing an ophthalmological condition involving RPE, most preferably AMD.

Description

[0080] The invention will now be described in more detail in the Examples below with reference to the following drawings in which:

[0081] FIG. 1 shows the results of a study in which the effect of sericin on gene expression was analysed by RNA microarray. HRPE cells were cultured for twelve days in DMEM with or without 1% sericin and were. The Venn diagram illustrates the number of transcripts with changed levels (fold change>1.5, nominal P<0.05). 209 and 229 transcripts were increased or reduced, respectively, while 14419 transcripts were unchanged. Transcripts with maximal log.sub.2 transformed signal values less than 5 were removed to filter for low and non-expressed genes.

[0082] FIG. 2 shows the results of a study analysing transcripts that changed upon addition of sericin. The NF-B complex was among the most highly activated (z-score: 4.597) upstream regulators. IPA revealed up-regulation of transcripts belonging to both the main and alternate NF-B pathway. Activation of the NF-B pathway was indicated by up-regulation of its components, including A20, Cot and NF-K132 p100.

[0083] FIG. 3 shows the results of pathway analyses which predict the downstream effects of culturing of HRPE cells with or without sericin. Ingenuity Pathway Analysis predicted a downstream effect compatible with increased viability and survival of human retinal pigment epithelial cells cultured in DMEM with 1% sericin compared to cells cultured without sericin. In the cell viability category (right center), 37 of 57 genes had an expression direction consistent with increased cell viability, yielding a Z-score of 2.8. In the cell survival category (left center), 38 of 61 genes had an expression direction consistent with increased cell survival, yielding a Z-score of 2.7. The upper two numbers below each symbols indicate P-values for cell survival or cell viability, respectively, while the lower number indicate fold change.

[0084] FIG. 4 shows photomicrographs showing human retinal pigment epithelial cells cultured for 14 days with or without 1% sericin using two different basal media. Magnification: 200. Photomicrographs are representative of eight samples.

[0085] FIG. 5 shows the ultrastrucure of HRPE cells cultured with sericin. Human retinal pigment epithelial HRPE cells were cultured in DMEM with 1% sericin for twelve days and then analysed by electron microscopy. (A) Scanning electron microscopy image showing the apical surface of a confluent layer of hexagonal HRPE cells with extensive microvilli (arrow). (B) Transmission electron microscopy image showing a polarized HRPE cell with apical tight junctions (black arrows), a basal nuclei and numerous melanosomes between stage I (non-melanized) and stage IV (melanized).

[0086] FIG. 6 shows the results of a study in which melanin content was measured by spectrophotometry. The bars show total protein adjusted relative levels of melanin after culturing HRPE cells for seven days with or without sericin as described. * P<0.05

[0087] FIG. 7 shows the results of a study in which the expression of the cellular retinaldehyde-binding protein (CRALBP) was measured by quantitative immunofluorescence in human retinal pigment epithelial cells cultured for 14 days in DMEM with or without 1% sericin and/or fetal bovine serum (FBS). (A) Photomicrographs show that CRALBP expression (red) was lowest in the DMEM/FBS group and highest in the DMEM/sericin group. Cell nuclei were counterstained with DAPI (blue). Magnification: 200. Photomicrographs are representative of four to eight samples. (B) Bar chart showing CRALBP expression in fold change relative to the DMEM alone group. N=4 to 8. Error bars: standard deviation. *P<0.01 compared to all other groups.

[0088] FIG. 8 shows the results of a study in which cell density and cell survival were measured by quantitative fluorescence in human retinal pigment epithelial cells cultured in DMEM with or without sericin or fetal bovine serum (FBS) for 14 days. (A) Bar chart showing cell density in fold change relative to the DMEM-group. Cell density was measured by counting DAPI-stained nuclei using ImageJ. N=8. Error bars: standard deviation. *P<0.001 compared to DMEM and DMEM with FBS; P=0.037 compared to DMEM with sericin and FBS. (B) Bar chart showing amount of dead cells in fold change relative to the DMEM-group. Cell death was measure by ethidium homodimer-1 (ETH-1) uptake by dead cells using a microplate reader. * P<0.001 compared to other two groups.

[0089] FIG. 9 shows the results of a study in which NF-B signalling was stimulated or inhibited in human retinal pigment epithelial cells. The NF-B activator inhibitor 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23) and the NF-B agonist phorbol-12-myristate-13-acetate (PMA) were used to assess whether activation of the NF-B pathway is necessary and sufficient for sericin-induced pigmentation of human retinal pigment epithelial (HRPE) cells following seven days of culture. (A) Photomicrograph showing pigment-containing HRPE cells following culture in DMEM with sericin. Magnification: 20. (B) Photomicrograph showing absence of pigment in HRPE cells following culture in DMEM with sericin and JSH-23. Magnification: 20. (C) Photomicrograph showing scarce pigment in HRPE cells following culture in DMEM with PMA. Magnification: 20.

EXAMPLES

Example 1

[0090] This study was carried out to determine whether a medium containing sericin is suitable for culture of RPE cells.

[0091] Materials and Methods

[0092] Normal HRPE and complete epithelial cell medium (EpiCM) were obtained from ScienCell Research Laboratories (San Diego, Calif.). Dulbecco's Modified Eagle's Medium (high glucose, with pyruvate; hereafter named DMEM), Minimal Essential Medium (-modification; MEM-), heat-inactivated fetal bovine serum (FBS), N1 growth supplement, taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, propidium iodide (P1) and 4,6-diamidino-2-phenylindole (DAPI) and 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23) were obtained from Sigma Aldrich (St Louis, Mo.). Nunclon surface 96-well plates, pipettes and other routine plastics came from VWR International (West Chester, Pa.). Mouse monoclonal anti-cellular retinaldehyde-binding protein (CRALBP; clone B2) was from Abcam (Cambridge, UK). Ethidium homodimer-1 (ETH-1) was from Invitrogen. Secondary Cy3-conjugated anti-mouse antibody was from Abcam. Ethidium homodimer 1 (EH-1) was obtained from Invitrogen (Carlsbad, Calif.). Soluene-350 was obtained from Perkin Elmer (Waltham, Mass.). Pierce BCA Protein Assay Kit was obtained from Bio-Rad (Hercules, Calif.).

[0093] Cell Culture

[0094] Third passage HRPE were seeded (7000 cells/cm.sup.2) in complete EpiCM on Nunclon surface 96-well plates and cultured under routine conditions of 95% air and 5% CO.sub.2 at 37 C. After two days, EpiCM was replaced with either: 1) DMEM with 1% FBS; 2) DMEM with 1% sericin; 3) DMEM without FBS or sericin; 4) MEM- with 1% FBS; 5) MEM- with 1% sericin; or 6) MEM- without FBS or sericin. All culture media based on DMEM were supplied with 10.000 U penicillin and 10 mg streptymocin at a final concentration of 1%. The MEM--based media were added taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, and N1 medium supplement, as described in Sonoda et al. (supra). The culture medium was changed every two to three days, and the HRPE were maintained in culture for a total of 14 days.

[0095] RNA Extraction and Microarray Hybridization

[0096] HRPE cells were cultured for 12 days and washed with PBS and lysed with QIAzol Lysis Reagent. The lysate was transferred to a microcentrifuge vial. Total RNA was then purified according to the manufacturer's protocol, and 100 ng of total RNA was processed with a GeneChip HT One-Cycle cDNA Synthesis Kit and a GeneChip HT IVT Labeling Kit (Affymetrix, Santa Clara, Calif.). Labeled and fragmented single stranded cDNAs were hybridized to the GeneChip Human Gene 1.0 ST Arrays (28,869 transcripts) (Affymetrix). Thereafter, the arrays were rinsed and stained using a FS-450 fluidics station (Affymetrix). Signal intensities were measured with a Hewlett Packard Gene Array Scanner 3000 7G (Hewlett Packard, Palo Alto, Calif.), and the scanned images were processed by the Affymetrix GeneChip Command Console (AGCC).The CEL files were imported into Partek Genomics Suite software (Partek, Inc. MO, USA). Robust microarray analysis (RMA) was applied for normalization. Gene transcripts with a maximal signal values less than 32 across all arrays were removed to filter for low and non-expressed genes, reducing the number of gene transcripts to 23190. Differentially expressed genes between groups were identified using one-way ANOVA analysis in Partek Genomics Suite Software. Clustering analysis was made using the same name module in a Partek Genomics Suite Software. Gene transcripts with maximal signal values of less than 5 were removed to filter for low and non-expressed genes, resulting in 14419 gene transcripts. For expression comparisons of different groups, profiles were compared using a 1-way ANOVA model. Data were presented as fold changes (FC) and P-values.

[0097] Microarray Data Analysis

[0098] Upstream analysis, pathway analysis and downstream predictions were performed using Ingenuity Pathways Analysis (IPA) (www.ingenuity.com).

[0099] Verification of Affymetrix data by RT-PCR

[0100] The differential gene expression data were validated for selected transcripts using TaqMan Gene Expression Assays and the Applied Biosystems ViiA 7 Real-Time PCR system (Applied Biosystem, Life technologies, Carlsbad, Calif.) (Table 1). Affymetrix analysis identified MAPRE1 and POLRSH as ideal for endogenous controls due to their stable expression across samples and groups. Briefly, 200 ng totaIRNA were reverse transcribed using gScript cDNA Super Mix (Quanta Biosciences, Gaithersburg, Md.) following the manufacturer's instructions. After completion of cDNA synthesis 1/10 of the first strand reaction were used for PCR amplification. 9 l of cDNA (diluted in H.sub.2O), 1 l of selected primer/probes TaqMan Gene Expression Assays (Life Technologies) and 10 l TagMan Universal Master Mix (Life Technologies) following the manufacturer's instructions. Normalized Ct values were calculated by subtracting the average Ct of the two endogenous controls from the Ct of the reference gene. Ct values were then calculated by subtracting normalized Ct values of the group containing cells cultured in DMEM with 1% sericin from the control group, which included cells cultured in DMEM without sericin. P-values were calculated using Student's t-Test in Microsoft Excel using delta Ct values. Normalized target gene expression levels (FC) were calculated using the formula: 2.sup.(Ct).

[0101] Light Microscopy

[0102] Cell morphology and presence of pigment was assessed after 14 days of culture by light microscopy at 200 magnification.

[0103] Transmission Electron Microscopy

[0104] HRPE cells cultured for 12 days in DMEM with 1% sericin were processed for transmission electronmicroscopy (TEM) analysis. In brief, ultrathin sections (60-70 nm thick) were cut on a Leica Ultracut Ultramicrotome (Leica, Wetzlar, Germany) and examined using a CM120 transmission electron microscope (Philips, Amsterdam, the Netherlands).

[0105] Scanning Electron Microscopy

[0106] HRPE cells cultured on glass coverslips in DMEM with 1% sericin for 12 days were used for scanning electron microscopy (SEM). Glutaraldehyde-fixed samples (n=3) were dehydrated in increasing ethanol concentrations and then dried according to the critical point method (Polaron E3100 Critical Point Drier, Polaron Equipment Ltd., Watford, UK) with CO.sub.2 as the transitional fluid. The specimens were attached to carbon stubs and coated with a 30 nm thick layer of platinum in a Polaron E5100 sputter coater before being photographed with an XL30 ESEM electron microscope (Philips, Amsterdam, The Netherlands).

[0107] Melanin Measurement by Spectrophotometry

[0108] Intracellular melanin was quantified in HRPE cultured in DMEM with and without 1% sericin for one week. Seven-day cultures were rinsed with PBS, and then re-suspended in 200 L of RIPA lysis buffer consisting of 25 mM Tris-HCL [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate dissolved in H.sub.2O. Part of the cell lysates were then incubated with Pierce BCA protein assay kit for 30 minutes at 37 C., and cooled to room temperature before measuring the absorbance at 562 nm using a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, Calif.). Soluene-350 was thereafter added to the remaining cell lysate (9:1). The cell lysate was subsequently incubated for 60 minutes at 80 C., before being centrifuged for 10 minutes at 8600 g. Solubilized melanin was measured at 490 nm on the microplate reader, and the concentration was adjusted by multiplication with protein levels.

[0109] Quantitative Immunofluorescence

[0110] Following 14 days of culture the cells were fixed in 100% methanol for 15 minutes and then washed three times with fresh PBS. Fixed cells were incubated for 45 minutes at room temperature in a blocking buffer consisting of 10% goat serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween-20, 0.05% sodium azide in PBS. Cells were then incubated overnight at 4 C. with the following antibody diluted in blocking buffer: anti-CRALBP (1:100), which targets a functional protein in the visual cycle and a marker for differentiated HRPE. The Cy3-conjugated secondary antibody, diluted in 0.2% PBST with 1% BSA, were incubated for one hour at room temperature. Negative control consisted of replacing the primary antibody with PBS. The cultures were thereafter rinsed three times in PBS and incubated with 1 g/mL DAPI in PBS to stain cell nuclei before a final wash with PBS.

[0111] Photomicrographs of the cultures were captured at 200 magnification using a Nikon Eclipse Ti fluorescence microscope with a DS-Qi1 black-and-white camera. The exposure length and gain was maintained at a constant level for all samples, and the fluorescence intensities of the Cy3 fluorochromes, which were conjugated to the secondary antibodies, were within the dynamic range of the camera.

[0112] Phenotype was quantified using ImageJ (National Institutes of Health, Bethesda, Md.) as described in e.g. Eidet et al. (Diagnostic Pathology, 2014. 9:92) with some modifications. In brief, mean fluorescence per cell was measured by enlarging regions of interest (ROI) created around the DAPI-stained nuclei to enclose the CRALBP-expressing cytosol. By using this method, we were able to normalize for differences in cell density in each photomicrograph.

[0113] Quantification of Cell Density

[0114] After 14 days in culture, the HRPE (n=8) were fixed with methanol, as described above. The cultures were then rinsed three times in PBS and incubated with 1 g/mL DAPI in PBS to stain cell nuclei before a final wash with PBS. Photomicrographs of the cultures were captured at 200 magnification with identical exposure length and gain. ImageJ was used to convert 16-bit images to binary images. The Analyze particles function in ImageJ was then used to automatically count cell nuclei per image.

[0115] Quantification of Cell Death

[0116] The amount of dead cells in the cultures after two weeks was quantified by incubating the samples with EH-1 for 30 minutes at 37 C. Ethidium homodimer-1 stains nuclei of dead cells red and its fluorescence was quantified by the microplate fluorometer with the excitation/emission filter pair 530/620. Background fluorescence, measured in wells incubated with EH-1-reagent, but without cells, was subtracted from all values before calculating mean fluorescence for the groups.

[0117] Statistical Analysis

[0118] One-way ANOVA with Tukey's post hoc pair-wise comparisons (SPSS ver. 21.0) was used to compare the groups. Data were expressed as meanstandard deviation, and values were considered significant if P<0.05.

[0119] Results

[0120] Microarray Analysis of HRPE Cultured with or without Sericin

[0121] Global Perspective. Gene expression differed considerably between the two culture groups, with a total of 438 significantly differentially regulated genes (fold change>1.5; P<0.05). In the sericin-supplemented group, 229 genes were down-regulated and 209 genes were up-regulated compared to the control (FIG. 1).

[0122] Upstream analysis. The top upstream regulators were tumor necrosis factor (TNF) (z-score: 5.008), interferon- (IFN-) (z-score: 4.623), the NF-B complex (z-score: 4.597) and interleukin-1(IL1) (z-score: 4.567). Pathway analysis of NF-B revealed up-regulation of transcripts belonging to both the main and alternate NF-B pathway (FIG. 2). Activation of the NF-B pathway was indicated by up-regulation of several of its members, including A20, Cot and NF-B2 p100 (Table 2).

[0123] Substantially Regulated Genes. The CXC motif chemokine 10 (CXCL10, also known as IP-10; interferon gamma-induced protein 10) was the most up-regulated gene in the dataset, with a 55.5-fold up-regulation in the sericin-supplemented group compared to the control (Table 3). Complement component 3 (C3) was the second most up-regulated gene in the sericin group, with a 34.7-fold up-regulation. The chemokine (CC motif) ligand 2 (CCL2) was up-regulated 21.6-fold in the sericin-supplemented group (Table 3).

[0124] Downstream Effects. The results obtained from the Ingenuity Pathway Analysis (IPA) predicted a downstream effect compatible with increased cell viability and survival in the sericin-supplemented group (FIG. 3). In the cell viability category, 37 of 57 genes had an expression direction consistent with increased cell viability, yielding a Z-score of 2.8. In the cell survival category, 38 of 61 genes had an expression direction consistent with increased cell survival, yielding a Z-score of 2.7.

[0125] RPE Related Gene Transcripts

[0126] Pigmentation. The (IPA) detected 12 differentially expressed genes related to cell pigmentation (Table 4). Eleven of the genes were up-regulated and one was down-regulated in HRPE cells cultured in DMEM with sericin compared to cells cultured in DMEM without sericin.

[0127] Visual Cycle. Several visual cycle genes were significantly up-regulated in HRPE cells cultured in DMEM with sericin compared to that of cells cultured in DMEM without sericin, while no visual cycle genes were significantly down-regulated (Table 5). Compared to cells cultured in DMEM without sericin, the cells that were cultured in DMEM with sericin displayed a 2.8-fold up-regulation of RPE65, a key isomerase of the visual cycle and an essential marker of RPE cell differentiation, and a 1.4-fold up-regulation of cellular retinaldehyde binding protein 1 (RLBP1; also known as CRALBP), which positions retinol for enzymatic turnover and thereby accelerates the process. Both retinol dehydrogenase 10 (RDH10) and retinol dehydrogenase 11 (RDH11) play complementary roles as 11-cis-retinol dehydrogenases in the visual cycle, and were up-regulated 1.9 and 1.6-fold, respectively, in cells cultured in DMEM with sericin compared to cells cultured in DMEM without sericin.

[0128] RT-PCR Validation

[0129] To validate the microarray results, NFKBIA, RPE65, CSF1, NFKBIZ and RGR transcripts were quantified by RT-PCR in HRPE cells cultured for twelve days in DMEM with or without 1% sericin. In Table 6, Ct values are transformed to fold change. All six transcripts were up-regulated in the Affymetrix and RT-PCR experiments (Table 6).

[0130] Microstructure following HRPE Culture with or without Sericin

[0131] Human retinal pigment epithelial cells were cultured with or without 1% sericin in two different basal media with or without FBS for 14 days before being assessed with light microscope for morphology and pigmentation (N=8). Widespread pigmentation was only seen in cells cultured with 1% sericin, irrespective of the basal medium used (MEM- or DMEM) (FIG. 1). Pigmented cells were rarely found in the groups that were cultivated without sericin. Typical cobblestone morphology with essentially hexagonal cells was seen in the samples cultured in DMEM with sericin, MEM- with sericin, MEM- with FBS, and MEM- alone (FIG. 4). Cell confluence was superior in DMEM with 1% sericin compared to DMEM alone and DMEM with 1% FBS. Hence, these results show that after 14 days in culture, only cells cultured in media supplied with sericin become pigmented and sericin appears to support HRPE cell confluence.

[0132] Ultrastructure following HRPE Culture with or without Sericin

[0133] Scanning electron microscopy of HRPE cultured for twelve days in DMEM with 1% sericin appeared tightly adjoined, hexagonal and had apical microvilli (FIG. 5A), hence demonstrating a differentiated ultrastructure. Transmission electron microscopy of HRPE cultured for twelve days in DMEM with 1% sericin contained melanosomes of all four stages following seven days of culture (FIG. 5B), thereby supporting the validity of the light microscopy experiments.

[0134] Melanin Quantification following HRPE Culture with or without Sericin

[0135] As the pigment melanin absorbs light at a specific wave length, measurement of pigment quantity is commonly performed by spectrophotometry. Following seven-day culture, spectrophotometry showed increased absorption at 562 nm in cells cultured in DMEM with 1% sericin (4.0 fold0.9; P=0.008) compared to cells cultured in DMEM without sericin (FIG. 6). Thus, the results further supports increased pigmentation in sericin-cultured cells.

[0136] Level of the CRALBP Protein following HRPE Culture with or without Sericin

[0137] To verify the presence and to assess the quantity of RPE-related proteins quantitative immunofluorescence was used to analyze the mean level of CRALBP, which is a protein integral to the visual cycle and associated with differentiated RPE. Following 14 days of culture, CRALBP was most abundant in cells cultured in DMEM with sericin, and present to a significantly higher degree than in cells cultured in DMEM alone, DMEM with FBS or DMEM with FBS and sericin (P<0.01) (FIG. 7). Retinal pigment epithelial cells cultured in DMEM with sericin and FBS also demonstrated a significantly higher expression of CRALBP compared to the DMEM alone and DMEM with FBS groups (P<0.01). Thus, quantitative immunofluorescence confirmed increased levels of CRALBP, as indicated by the microarray results.

[0138] HRPE Cell Density and Cell Death following Serum Starvation

[0139] As the microarray results predicted increased viability of culturing cells in DMEM with sericin compared to DMEM without sericin, cell density and cell death after 14 days of cultivation with or without sericin was quantified by counting DAPI-stained cell nuclei and by microplate fluorometer measurements of EH-1, the latter which is indicative of dead cells. DMEM with sericin yielded the highest cell density and significantly higher density than DMEM alone (P<0.001), DMEM with FBS (P<0.001) and DMEM with sericin and FBS (P=0.037) (FIG. 8).

[0140] Cultured HRPE were assayed with EH-1 to quantify the number of dead cells following 14 days of culture (N=8). The highest number of dead cells was obtained when using DMEM alone (FIG. 8B). Cells cultured in this medium demonstrated a significantly higher amount of dead cells than when using DMEM with sericin or DMEM with FBS (P<0.001). Thus, the cell density and cell death results supported the microarray prediction of increased viability when culturing HRPE in DMEM with sericin.

[0141] NF-B Pathway and Melanization of Cultured HRPE Cells

[0142] The NF-B-inhibitor JSH-23 specifically inhibits nuclear translocation and activation of NF-B. The addition of JSH-23 to a culture medium consisting of DMEM with 1% sericin completely prevented development of pigmented cells visible by light microscopy following seven days of culture, as opposed to control cells cultured in DMEM with sericin, but without JSH-23 (FIGS. 9A and B). However, stimulation of the NF-B pathway by adding PMA, a NF-B agonist, to a culture medium consisting of DMEM without sericin did not induce melanization (FIG. 9C). Thus, NF-B activation appears to be necessary, but not sufficient, for sericin-induced melanization of HRPE.

[0143] Discussion

[0144] In the current study, sericin induced pigmentation of cultured HRPE by NF-B pathway activation while still preserving cell viability and improving RPE differentiation, as indicated by pathway analysis of Affymetrix microarray, micro and ultra-structural studies, spectrophotometry, quantitative immunofluorescence and viability assays.

[0145] The NF-B pathway was, alongside TNF, IFN- and IL1, one of the top upstream sericin-induced regulators in this study. The NF-B pathway is involved in multiple cellular processes, including inflammation and immunity. NF-B is also part of the TNF pathway and is regulated by IL1. A link between inflammatory cytokines, including IL1 and TNF-, and induction of pigmentation in chick RPE cells has been reported. Interestingly, IFN- activation has been related to hypopigmentation in skin melanocytes. In our study, the addition of the NF-B activator inhibitor JSH-23 prevented pigmentation in sericin-cultured cells and thereby confirmed the role of NF-B in pigmentation of HRPE, whereas the relatively scarce pigmentation achieved with the NF-B agonist PMA alone suggested that sericin promotes pigmentation, albeit not exclusively, by activating the NF-B pathway.

[0146] IPA identified several genes that are potentially linked to sericin-induced pigmentation, including ABCA4, PROM1, C10orf11, SLC24A5, TGF2 and IRF1, which were all up-regulated by sericin. Of these genes, mutations in ABCA4 have been related to Stargardt disease and retinitis pigmentosa (RP), with associated pigment disturbances. PROM1 is also related to maculopathy, as mutations in this gene may cause macular degeneration, including dominant bull's eye maculopathy. Mutations in C10orf11 have been shown to decrease pigmentation of melanocytes and lead to human albinism. Interestingly, down-regulation of SLC24A5 causes reduced melanin content in chick RPE. Furthermore, SLC24A5 has also been related to melanin content in skin melanocytes and the gene product of SLC24A5 localizes to intracellular membranes, including melanosomes. Inhibition of TGF2 has been reported to suppress melanogenesis in human melanoma cells. Surprisingly, up-regulation of IRF1 has been associated with hypo-pigmentation in skin melanocytes. Thus, of all the genes identified by IPA as being associated with sericin-induced pigmentation in this study, C10rf11, SLC24A5 and TGF2 appear to be the most promising.

[0147] To our knowledge there are no studies on the effects of sericin on RPE melanogenesis for direct comparison to our study. However, previous reports have demonstrated that sericin inhibits tyrosinase, which is the main rate-limiting melanogenesis enzyme, thus the induction of pigmentation by sericin in our study is unexpected. Tyrosinase catalyses the formation of dihydroxyphenylalanine (L-DOPA) from L-tyrosine. L-DOPA is subsequently converted to melanin, aided by tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2). Tyrosinase is inhibited by acidic conditions, including those resulting from high metabolic activity. The microarray data did not reveal any significant effect of sericin on the tyrosinase transcript TYR, or on TRP-1 and TRP-2, thereby suggesting that sericin's effect on pigmentation is unrelated to regulation of these genes. The production of melanin is a complex process, however, involving several steps where sericin could have a stimulating role that fully compensates for its acclaimed tyrosinase-inhibiting effect.

[0148] Pigmentation was almost exclusively seen in cells that had been cultured in sericin-supplemented basal media (either MEM- or DMEM). Hexagonal cobblestone morphology was also achieved when using either basal media supplemented with sericin. After culturing the cell line ARPE-19 for 98 days in DMEM with FBS, Ahmado and co-workers demonstrated pigmented and hexagonal cells Ahmado et al. 2011 (supra). To our knowledge, this medium has not been used for normal RPE. The MEM--based medium, however, has been shown to induce pigmentation in normal HRPE cells in study by Sonoda et al. 2009 (supra). In contrast to our study, the HRPE in their study was pigmented upon start of culture and, after initial depigmentation, became repigmented after 14 days. Both MEM- and DMEM supplemented with FBS are known to induce differentiation of RPE cells in prolonged culture (>3 weeks/months). Our culture time of seven to 14 days may, therefore, have been too short time for widespread melanogenesis to occur in these media.

[0149] In RPE, pigment can be seen within vesicles called melanosomes, which can be divided into four stages of development based on ultrastructure. While stage I and II melanosomes are amelanotic, stage III and IV melanosomes are partially, and fully, pigmented, respectively. HRPE cultured in DMEM containing sericin displayed melanosomes of all four stages following seven days of culture, which suggests that the process of melanogenesis was ongoing.

[0150] The tight junction barrier is involved in creating a polarized epithelium and is necessary for maintaining an apical-basal concentration gradient across the RPE. Thus, as shown by TEM in the current study, sericin enabled the development of a polarized RPE, as indicated by presence of apical tight junctions and basal cell nuclei.

[0151] A20, CXCL10, C3 and CCL2 were among the top five sericin-induced genes in this study. A20 was also the top up-regulated gene in the NF-B pathway. It is anti-inflammatory and prevents NF-B and TNF-mediated apoptosis (Beyaert et al. Biochemical pharmacology. 2000; 60(8):1143-51). A20 is induced by TNF and inhibits the NF-B pathway by de-ubiquitination and ubiquitination of the TNF receptor-interacting protein (Heyninck et al. Trends in biochemical sciences. 2005; 30(1):1-4). Zinc supplementation in human AMD patients, which up-regulates A20 (Prasad et al. Free radical biology & medicine. 2004; 37(8):1182-90), has been shown to be associated with decreased risk of developing advanced AMD or neo-vascular AMD during a 10-year follow-up (Chew et al. Ophthalmology. 2013; 120(8):1604-11 e4). In rats, A20 (TNFAIP3) was identified as a candidate gene for development of retinopathy (Korbolina et al. BMC genomics. 2014; 15 Suppl 12:S3). The CXCL10 protein is a potent inhibitor of angiogenesis and an antitumor agent causing tumor necrosis. C3 is commonly found in drusen of AMD patients, and the presence of C3 is critical for protection of the retina. Absence of C3 expression has deleterious effects on the retinal structure and leads to progressive retinal degeneration. C3 can also initiate angiogenesis, thereby opposing the anti-angiogenic effect of CXCL10. CCL2 contributes in maintaining normal RPE morphology, and lack of the gene leads to RPE cell loss and stress.

[0152] Inclusion of sericin in the culture medium leads to up-regulation of several genes related to the visual cycle, including RPE65, RDH10 and CRALBP. Retinal diseases can result from mutations or malfunction of key proteins in the visual cycle, in which the RPE serves as a critical component. The RDH10 is essential for synthesis of embryonic retinoic acid and therefore for limb, craniofacial and organ development. RPE65 is one of the key markers of RPE cells, and responsible for light-independent conversion of all-trans-retinyl esters into 11-cis-retinol. CRALBP, a marker of HRPE differentiation that is involved in retinol recycling, was increased by sericin, as demonstrated by microarray and immunofluorescence. Thus, sericin increases maturation of HRPE by both promoting melanogenesis and the visual cycle.

[0153] Viability analyses were performed to investigate whether the sericin-induced up-regulation of several inflammatory cytokines was accompanied with increased cell death. To reduce the effect of cell proliferation on cell density, the measurements of cell density were performed after 14 days of post-confluent culture with or without sericin or FBS. Sericin appeared to preserve cell density under serum-free conditions, and resulted in higher cell density than when adding either FBS or a combination of FBS and sericin to DMEM. Corroborating experiments with ETH-1 demonstrated that sericin promotes cell survival, which is in line with the downstream prediction made by IPA. Our results are further supported by a study demonstrating that sericin protects against cell death following acute serum-deprivation and studies showing that FBS can be replaced by sericin in cryopreservation media without compromising viability Sasaki et al. (Biotechnology and Applied Biochemistry 2005; 42(Pt 2):183-8) and Verdanova et al. (Biopreservation and Biobanking 2014; 12(2):99-105). The increased survival of hRPE upon stimulation with sericin may be related to up-regulation of A20 (TNFAIP3), which inhibits TNF-induced apoptosis, or up-regulation of anti-oxidant genes, including the pigmentation-related gene SOD2. Downstream analysis by IPA also predicted a relationship between up-regulation of TNFAIP3 and SOD2 and increased cell viability and cell survival. In addition, a direct reactive oxygen species-scavenging effect of sericin has been reported elsewhere (Chlapanidas et al. International journal of biological macromolecules. 2013; 58:47-56). In RPE, inflammatory cytokines, such as IFN- and TNF-, have been shown to induce SOD2, which promotes cell survival in the presence of oxidative stress (Juel et al. PloS one. 2013;8(5):e64619). Thus, even though sericin promoted augmented expression of the inflammatory NF-B pathway, cell survival was increased, possibly by the concomitant up-regulation of anti-apoptotic and anti-oxidant genes.

[0154] In conclusion, sericin promotes pigmentation of cultured HRPE by activating the NF-B pathway. Sericin's potential role in culture protocols for rapid differentiation of RPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

TABLE-US-00001 TABLE 1 Taqman Assays Gene symbol Assay ID RPE65 Hs01071462_m1 RGR Hs00173619_m1 NFKBIA Hs00355671_g1 NFKBIZ Hs00230071_m1 CSF1 Hs00174164_m1 POLR3H Hs00978014_m1 MAPRE1 Hs01121102_g1

TABLE-US-00002 TABLE 2 Differentially Expressed Genes in the NFB-pathway in HRPE Cultured in DMEM with Sericin Gene Symbol Gene Name Affymetrix ID FC P-value A20 Tumor necrosis factor, alpha- 17012946 10.4 8.7E05 induced protein 3 Cot Mitogen-activated protein kinase 16703642 2.5 1.1E02 kinase kinase 8 NF-B2 Nuclear factor of kappa light 16708623 2.1 3.3E05 p100 polypeptide gene enhancer in B- cells 2 (p49/p100) BAFF Tumor necrosis factor (ligand) 16776339 2.1 1.1E03 superfamily, member 13b NF-B1 Nuclear factor of kappa light 16969300 2.0 2.3E04 polypeptide gene enhancer in B- cells 1 ABIN-1 Tumor necrosis factor, alpha- 17012946 1.8 8.7E05 induced protein 3 RelB TNFAIP3 interacting protein 1 17001763 1.6 3.5E04 EGF V-rel avian reticuloendotheliosis 16863168 1.6 9.0E04 viral oncogene homolog B MEKK1 Epidermal growth factor 16969729 1.4 1.1E02 TGF- Mitogen-activated protein kinase 16984945 1.2 1.5E02 kinase kinase 1, E3 ubiquitin protein ligase TAB1 Transforming growth factor, alpha 16898788 1.1 4.5E02 HRPE = human retinal pigment epithelial cells; DMEM = dulbecco modified eagle medium; FC = fold change

TABLE-US-00003 TABLE 3 Top Ten Up- and Down-regulated Genes in HRPE Cultured in DMEM with Sericin Gene Affymetrix Symbol Gene Name ID FC P-value CXCL10 Chemokine (C-X-C motif) ligand 10 16977052 55.5 4.2E06 C3 Complement component 3 16867784 34.7 1.2E06 CCL2 Chemokine (C-C motif) ligand 2 16833204 21.6 9.3E07 IL6 Interleukin 6 17044177 15.6 8.8E06 TNFAIP3 Tumor necrosis factor, alpha- 17012946 10.4 8.7E05 induced protein 3 ICAM1 Intercellular adhesion molecule 1 16858137 9.1 2.0E06 PTX3 Pentraxin 3, long 16947357 8.0 1.4E04 ADA Adenosine deaminase 16919466 7.7 2.6E06 EDNRB Endothelin receptor type B 16779958 7.5 1.6E05 CNTNAP1 Contactin associated protein 1 16834409 7.3 5.3E06 CLGN Calmegin 16980051 5.3 4.0E06 ADAM28 ADAM metallopeptidase domain 28 17066921 5.5 2.8E05 LUZP2 Leucine zipper protein 2 16722987 5.6 8.7E05 VCAN Versican 16986913 6.4 2.6E05 MFAP4 Microfibrillar-associated protein 4 16842266 6.6 3.9E06 LRRC15 Leucine rich repeat containing 15 16962911 6.9 3.2E04 ST8SIA4 ST8 alpha-N-acetyl-neuraminide 16998532 6.9 7.3E06 alpha-2,8-sialyltransferase 4 COL14A1 Collagen, type XIV, alpha 1 17072162 9.2 4.5E07 SMOC2 SPARC related modular calcium 17014798 9.8 2.8E08 binding 2 GRIK3 Glutamate receptor, ionotropic, 16685330 16.4 5.3E06 kainate 3 HRPE = human retinal pigment epithelial cells; DMEM = dulbecco modified eagle medium; FC = fold change

TABLE-US-00004 TABLE 4 Differentially Expressed Pigmentation-associated Genes in HRPE Cultured in DMEM with Sericin Gene Affymetrix Symbol Gene Name ID FC P-value IL6 Interleukin 6 17044177 15.6 8.8E06 EDNRB Endothelin receptor type B 16779958 7.5 1.6E05 SOD2 Superoxide dismutase 2, 17025267 4.6 1.6E05 mitochondrial ABCA4 ATP-binding cassette, sub- 16689764 3.8 2.8E05 family A (ABC1), member 4 C10orf11 Chromosome 10 open 16706350 3.1 1.1E05 reading frame 11 IRF1 Interferon regulatory 16999776 3.0 1.0E03 factor 1 PROM1 Prominin 1 16974534 2.9 2.6E05 INSIG1 Insulin induced gene 1 17053892 2.9 1.2E04 SLC24A5 Solute carrier family 24, 16800764 2.2 1.8E04 member 5 SOD3 Superoxide dismutase 3, 16965519 2.1 4.1E04 extracellular TGF2 Transglutaminase 2 16919158 2.0 6.9E04 HELLS Helicase, lymphoid-specific 16707695 2.0 3.4E03 HRPE = human retinal pigment epithelial cells; DMEM = dulbecco modified eagle medium; FC = fold change

TABLE-US-00005 TABLE 5 Differentially Expressed Visual Cycle-associated Genes in HRPE Cultured in DMEM with Sericin Gene Affymetrix Symbol Gene Name ID FC P-value RPE65 Retinal pigment epithelium- 16688370 2.8 2.7E05 specific protein 65 kDa RDH10 Retinol dehydrogenase 17070013 1.9 1.7E05 10 (all-trans) RDH11 Retinol dehydrogenase 11 16794064 1.7 3.7E04 (all-trans/9-cis/11-cis) RLBP1 Retinaldehyde binding 16813062 1.4 8.0E04 (CRALBP) protein 1 HRPE = human retinal pigment epithelial cells; DMEM = dulbecco modified eagle medium; FC = fold change

TABLE-US-00006 TABLE 6 Verification of Affymetrix data by RT PCR Gene RT PCR Affymetrix symbol Gene Name FC P-value FC P-value NFKBIA Nuclear factor of kappa 12.9 4.0E06 6.9 2.0E03 light polypeptide gene enhancer in B-cells inhibitor, alpha RPE65 Retinal pigment 4.3 1.8E07 2.8 2.7E05 epithelium- specific protein 65 kDa CSF1 Colony stimulating factor 6.8 1.8E04 2.8 1.0E02 1 (macrophage) NFKBIZ Nuclear factor of kappa 5.1 2.0E03 2.6 2.0E02 light polypeptide gene enhancer in B-cells inhibitor, zeta RGR Retinal G protein 3.6 5.4E05 2.3 2.0E03 coupled receptor The table shows average fold change (increase) in mRNA levels upon incubation of primary RPE cells with sericin for 12 days as compared to controls. Data are average from triplicates with MAPRE1 and POLR5H as endogenous controls. RT PCR = real time polymerase chain reaction; FC = fold change