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

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          Abstract

          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)β-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.

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          Most cited references 52

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          Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies

          The Lancet, 385(9967), 509-516
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            Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

            Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media. Copyright 2010 Elsevier Ltd. All rights reserved.
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              Treatment of Macular Degeneration Using Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Preliminary Results in Asian Patients

              Summary Embryonic stem cells hold great promise for various diseases because of their unlimited capacity for self-renewal and ability to differentiate into any cell type in the body. However, despite over 3 decades of research, there have been no reports on the safety and potential efficacy of pluripotent stem cell progeny in Asian patients with any disease. Here, we report the safety and tolerability of subretinal transplantation of human embryonic-stem-cell (hESC)-derived retinal pigment epithelium in four Asian patients: two with dry age-related macular degeneration and two with Stargardt macular dystrophy. They were followed for 1 year. There was no evidence of adverse proliferation, tumorigenicity, ectopic tissue formation, or other serious safety issues related to the transplanted cells. Visual acuity improved 9–19 letters in three patients and remained stable (+1 letter) in one patient. The results confirmed that hESC-derived cells could serve as a potentially safe new source for regenerative medicine.
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                Author and article information

                Journal
                Transl Vis Sci Technol
                Transl Vis Sci Technol
                tvst
                tvst
                tvst
                Translational Vision Science & Technology
                The Association for Research in Vision and Ophthalmology
                2164-2591
                22 February 2016
                February 2016
                : 5
                : 1
                Affiliations
                [1 ]Institute of Ophthalmology and Visual Science Rutgers, New Jersey Medical School, Newark, NJ, USA
                [2 ]Department of Biochemistry and Molecular Biology, Center for Advanced Proteomics Research, Neuroproteomics Core Facility, Rutgers, New Jersey Medical School, Newark, NJ, USA
                Author notes
                Correspondence: Marco A. Zarbin, Institute of Ophthalmology and Visual Science, 90 Bergen Street, Newark, NJ 07101-1709, USA; zarbin@ 123456earthlink.net
                Article
                tvst-05-01-10 MS#: TVST-15-0277
                10.1167/tvst.5.1.8
                4771074
                26933521
                b3490bfe-58fe-4fc8-993f-826e7fd53fc6

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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