In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells.
OBJECTIVE
To determine whether primate embryonic stem (ES) cell-derived pigment epithelial cells (ESPEs) have the properties and functions of retinal pigment epithelial (RPE) cells in vitro and in vivo.


METHODS
Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by coculturing them with PA6 stromal cells in a differentiating medium. The expanded, single-layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT-PCR, Western blot, and immunocytochemical analyses. The ESPEs were transplanted into the subretinal space of 4-week-old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks after grafting. The effect of the ESPE graft on the visual function of RCS rats was estimated by optokinetic reflex.


RESULTS
The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: ZO-1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histologic analyses and behavioral tests.


CONCLUSIONS
The ESPEs had morphologic and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction.
Haruta
Masatoshi
M
Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan.
Sasai
Yoshiki
Y
Kawasaki
Hiroshi
H
Amemiya
Kaori
K
Ooto
Sotaro
S
Kitada
Masaaki
M
Suemori
Hirofumi
H
Nakatsuji
Norio
N
Ide
Chizuka
C
Honda
Yoshihito
Y
Takahashi
Masayo
M
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D005136:Eye Proteins; D019084:Fluorescent Antibody Technique, Indirect; D008252:Macaca fascicularis; D008565:Membrane Proteins / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism / Q000473:pathology; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000502:physiology / Q000637:transplantation*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D011922:Rats, Mutant Strains; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D012162:Retinal Degeneration / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D062826:Zonula Occludens-1 Protein; D000076202:c-Mer Tyrosine Kinase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D015415:Biomarkers; D002352:Carrier Proteins; D005136:Eye Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020794:Receptor Protein-Tyrosine Kinases; D062826:Zonula Occludens-1 Protein; D000076202:c-Mer Tyrosine Kinase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C020932:11-cis-retinal-binding protein; C569044:Tjp1 protein, rat; C490765:Mertk protein, rat