Two Bioactive Molecular Weight Fractions of a Conditioned Medium Enhance RPE Cell Survival on Age-Related Macular Degeneration and Aged Bruch's Membrane.

PURPOSE: To characterize molecular weight fractions of bovine corneal endothelial cell conditioned medium (CM) supporting retinal pigment epithelium (RPE) cell survival on aged and age-related macular degeneration (AMD) Bruch's membrane. METHODS: CM was subject to size separation using centrifugal filters. Retentate and filtrate fractions were tested for bioactivity by analyzing RPE survival on submacular Bruch's membrane of aged and AMD donor eyes and behavior on collagen I-coated tissue culture wells. Protein and peptide composition of active fractions was determined by mass spectrometry. RESULTS: Two bioactive fractions, 3-kDa filtrate and a 10-50-kDa fraction, were necessary for RPE survival on aged and AMD Bruch's membrane. The 3-kDa filtrate, but not the 10-50-kDa fraction, supported RPE growth on collagen 1-coated tissue culture plates. Mass spectrometry of the 10-50-kDa fraction identified 175 extracellular proteins, including growth factors and extracellular matrix molecules. Transforming growth factor (TGF)ß-2 was identified as unique to active CM. Peptides representing 29 unique proteins were identified in the 3-KDa filtrate. CONCLUSIONS: These results indicate there is a minimum of two bioactive molecules in CM, one found in the 3-kDa filtrate and one in the 10-50-kDa fraction, and that bioactive molecules in both fractions must be present to ensure RPE survival on Bruch's membrane. Mass spectrometry analysis suggested proteins to test in future studies to identify proteins that may contribute to CM bioactivity. TRANSLATIONAL RELEVANCE: Results of this study are the first steps in development of an adjunct to cell-based therapy to ensure cell transplant survival and functionality in AMD patients.

Biochemical restoration of aged human Bruch's membrane: experimental studies to improve retinal pigment epithelium transplant survival and differentiation.

Suspensions of human embryonic stem cell-derived retinal pigment epithelium (hES-RPE) and human fetal RPE resurface aged and age-related macular degeneration (AMD) Bruch's membrane to a limited degree at day 21 in organ culture. Survival and differentiation of hES-RPE and human fetal RPE on aged or AMD Bruch's membrane are enhanced greatly (200%) if a biologically synthesized extracellular matrix (bovine corneal endothelial cell extracellular matrix) is laid down on Bruch's membrane prior to transplantation. Transplanted RPE survival is enhanced even more (400-1,000%) if Bruch's membrane is treated with bovine corneal endothelial cell-conditioned medium during organ culture of hES-RPE or fetal RPE on aged or AMD Bruch's membrane. Future efforts are focused on identifying the bioactive components of bovine corneal endothelial cell-conditioned medium, so that this material can be reconstituted for clinical use as an adjunct to improve RPE transplant survival and differentiation in AMD eyes.

A method to enhance cell survival on Bruch's membrane in eyes affected by age and age-related macular degeneration.

PURPOSE: To determine whether conditioned medium (CM) derived from bovine corneal endothelial cells (BCECs) can support transplanted cells on aged and age-related macular degeneration (AMD) Bruch's membrane (BM). METHODS: Retinal pigment epithelium (RPE) cells derived from human embryonic stem cells (hES-RPE) and cultured fetal and aged adult RPE were seeded onto the inner collagenous layer of submacular BM-choroid-sclera explants generated from aged and AMD human donor eyes. Paired explants were cultured in BCEC-CM or CM vehicle. To assess cell behavior after attachment to BM was established, explants were harvested after 21 days in culture. To assess whether sustained exposure to BCEC-CM was necessary for improved cell survival on BM, short exposure to BCEC-CM (3, 7, 14 days) was compared with 21-day exposure. Explants were harvested and evaluated by scanning electron and light microscopy. Extracellular matrix (ECM) deposition after exposure to BCEC-CM was evaluated following RPE cell removal after day 21 on tissue culture dishes or on BM. RESULTS: BCEC-CM significantly enhanced hES-RPE, fetal RPE, and aged adult RPE survival on BM, regardless of submacular pathology. Although shorter BCEC-CM exposure times showed significant improvement in cell survival compared with culture in CM vehicle, longer BCEC-CM exposure times were more effective. BCEC-CM increased RPE ECM deposition on tissue culture plastic and on BM. CONCLUSIONS: The results of this study indicate that RPE survival is possible on AMD BM and offer a method that could be developed for enhancing transplanted cell survival on AMD BM. Increased ECM deposition may account for improved cell survival after culture in BCEC-CM.

Comparison of FRPE and human embryonic stem cell-derived RPE behavior on aged human Bruch's membrane.

PURPOSE: To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors. METHODS: hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. RESULTS: hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. CONCLUSIONS: Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.

Cell-deposited matrix improves retinal pigment epithelium survival on aged submacular human Bruch's membrane.

PURPOSE: To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. METHODS: Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. RESULTS: The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. CONCLUSIONS: Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes.

Short-term study of retinal pigment epithelium sheet transplants onto Bruch's membrane.

The purpose of this study is to investigate the survival and behaviour of retinal pigment epithelium sheets transplanted onto hydraulically debrided Bruch's membrane. Uncultured retinal pigment epithelium sheets obtained from male cats and sandwiched between two gelatin sheets were transplanted onto the tapetal area of female cats after native retinal pigment epithelium was debrided. For controls, the gelatin carrier was transplanted after debridement. Each transplant or control specimen was analyzed histologically and immunohistochemically. Transplanted male retinal pigment epithelial cells were identified by in situ labelling of the cat Y chromosome. Over half of the transplants appeared as retinal pigment epithelium multilayers in the subretinal space. Retinal pigment epithelium pigment dispersion into the subretinal space was seen in most of the transplants, and retinal pigment epithelium pigment infiltration into the neural retina was seen in all 7-day survival transplants. A few condensed darkly stained retinal pigment epithelium nuclei and Terminal Transferase dUTP Nick End Labelling-positive retinal pigment epithelium cells were observed in all transplants. Cellular retinaldehyde-binding protein was present up to day-7 in most transplanted RPE cells. In both transplant and control specimens, the antibody against the Ki-67 nuclear antigen labelled a few retinal pigment epithelium cells at day-3. Terminal Transferase dUTP Nick End Labelling-positive outer nuclear layer nuclei were most frequently observed at day-1 but were much less frequent at day-3 in both transplants and controls. The survival and effectiveness of retinal pigment epithelium sheet transplants appeared similar to the retinal pigment epithelium microaggregates transplants conducted previously in this model.