Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay.
OBJECTIVE
To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells.


METHODS
HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT-PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology.


RESULTS
HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures.


CONCLUSIONS
The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.
Carr
Amanda-Jayne
AJ
Institute of Ophthalmology, University College London, London, UK. a.carr@ucl.ac.uk
Vugler
Anthony
A
Lawrence
Jean
J
Chen
Li Li
LL
Ahmado
Ahmed
A
Chen
Fred K
FK
Semo
Ma'ayan
M
Gias
Carlos
C
da Cruz
Lyndon
L
Moore
Harry D
HD
Walsh
James
J
Coffey
Peter J
PJ
D000704:Analysis of Variance; D000818:Animals; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D010587:Phagocytosis / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000502:physiology / Q000648:ultrastructure; D055214:Retinal Photoreceptor Cell Outer Segment / Q000502:physiology*; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D013552:Swine; D000076202:c-Mer Tyrosine Kinase
D005136:Eye Proteins; D011518:Proto-Oncogene Proteins; C097072:MERTK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase