BackgroundAge-related macular degeneration (AMD) is a leading cause of blindness among the elderly characterized by retinal pigment epithelium (RPE) degeneration with accumulation of abnormal intracellular deposits (lipofuscin) and photoreceptor death. RPE is vital for the retina and integrity of photoreceptors through its phagocytic function which is closely linked to formation of lipofuscin through daily phagocytosis of discarded photoreceptor outer segments (POS). Although phagocytosis has been implicated in AMD, it has not been directly shown to be altered in AMD. RPE phagocytic defect was previously shown to be rescued by subretinal injection of human umbilical tissue derived cells (hUTC) in a rodent model of retinal degeneration (RCS rat) through receptor tyrosine kinase (RTK) ligands and bridge molecules. Here, we examined RPE phagocytic function directly in the RPE from AMD patients and the ability and mechanisms of hUTC to affect phagocytosis in the human RPE.MethodsHuman RPE was isolated from the post-mortem eyes of normal and AMD-affected subjects and cultured. RPE phagocytic function was measured in vitro using isolated POS. The effects of hUTC conditioned media, recombinant RTK ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as bridge molecules milk-fat-globule-EGF-factor 8 (MFG-E8), thrombospondin (TSP)-1, and TSP-2 on phagocytosis were also examined in phagocytosis assays using isolated POS. RNA was isolated from normal and AMD RPE treated with hUTC conditioned media and subjected to transcriptome profiling by RNA-Seq and computational analyses.ResultsRPE phagocytosis, while showing a moderate decline with age, was significantly reduced in AMD RPE, more than expected for age. hUTC conditioned media stimulated phagocytosis in the normal human RPE and significantly rescued the phagocytic dysfunction in the AMD RPE. RTK ligands and bridge molecules duplicated the rescue effect. Moreover, multiple molecular pathways involving phagocytosis, apoptosis, oxidative stress, inflammation, immune activation, and cholesterol transport were affected by hUTC in the RPE.ConclusionsWe demonstrated for the first time RPE phagocytic dysfunction in AMD, highlighting its likely importance in AMD, and the ability of hUTC to correct this dysfunction, providing insights into the therapeutic potential of hUTC for AMD.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1434-6) contains supplementary material, which is available to authorized users.

Retinal pigment epithelium (RPE) cells perform many functions crucial for retinal preservation and vision. RPE cell dysfunction results in various retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD). Currently, there are no effective treatments for retinal degeneration except for a small percentage of individuals with exudative AMD. Cell therapies targeting RPE cells are being developed in the clinic for the treatment of retinal degeneration. Subretinal injection of human umbilical tissue-derived cells (hUTC) in the Royal College of Surgeons (RCS) rat model of retinal degeneration was shown to preserve photoreceptors and visual function. However, the precise mechanism remains unclear. Here, we demonstrate that hUTC rescue phagocytic dysfunction in RCS RPE cells in vitro. hUTC secrete receptor tyrosine kinase (RTK) ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as opsonizing bridge molecules milk-fat-globule-epidermal growth factor 8 (MFG-E8), growth arrest-specific 6 (Gas6), thrombospondin (TSP)-1, and TSP-2. The effect of hUTC on phagocytosis rescue in vitro is mimicked by recombinant human proteins of these factors and is abolished by siRNAtargeted gene silencing in hUTC. The bridge molecules secreted from hUTC bind to the photoreceptor outer segments and facilitate their ingestion by the RPE. This study elucidates novel cellular mechanisms for the repair of RPE function in retinal degeneration through RTK ligands and bridge molecules, and demonstrates the potential of using hUTC for the treatment of retinal degenerative diseases. STEM CELLS 2016;34:367-379 SIGNIFICANCE STATEMENTRetinal degeneration is a leading cause of blindness. There is currently no effective treatment. Retinal pigment epithelium (RPE) play a pivotal role in retinal preservation. The phagocytic function of the RPE is crucial for the integrity of the light-sensing photoreceptors, thus vision. We previously reported that subretinal injection of human umbilical tissue-derived cells (hUTC) in a rodent model of retinal degeneration preserved photoreceptors and vision with an unknown mechanism. Here, we report that hUTC can rescue RPE phagocytic dysfunction through the release of receptor tyrosine kinase ligands and bridge molecules. This study identifies novel mechanisms of a cell-based therapy for correcting the RPE dysfunction in retinal degeneration, suggesting that hUTC may provide utility in treating patients with retinal degenerative diseases, such as AMD and retinitis pigmentosa.

Hamm-Alvarez SF. Reduced expression of VEGF-A in human retinal pigment epithelial cells and human muller cells following CRISPR-Cas9 ribonucleoprotein-mediated gene disruption.