In vitro and in vivo characterization of iris pigment epithelial cells cultured on amniotic membranes.
OBJECTIVE
To determine whether human amniotic membranes (AMs) can induce human and rat iris pigment epithelial (IPE) cells grown on them to develop characteristics of RPE cells in situ better than IPE cells grown on plastic plates, and to determine whether subretinal transplantation of IPE cell sheets grown on AMs can protect photoreceptor cells in dystrophic Royal College of Surgeons (RCS) rats.


METHODS
IPE cells from humans and Long-Evans rats were cultured on the basement membrane side of dispase-treated AMs. Two weeks after seeding, ultrastructural changes were evaluated by transmission electron microscopy, and the level of expression of several genes present in differentiated retinal pigment epithelial (RPE) cells was determined by real time PCR and western blotting. IPE cell sheets cultured on AMs were transplanted into the subretinal space of 4-week-old RCS rats, and eyes were analyzed histologically 12 weeks after grafting.


RESULTS
IPE cells cultured on AMs showed ultrastructural features like intercellular junctions, similar to RPE cells in situ. IPE cells grown on AMs had a greater upregulation in the expression of genes important for the function of differentiated RPE cells (e.g., pigment epithelium-derived factor [PEDF], RPE65, bestrophin, VEGF, and BDNF) than IPE cells grown on plastic plates. The number of photoreceptors present in RCS rats after subretinal transplantation of IPE cell sheets grown on AMs was significantly higher than that of sham injected rats and rats receiving transplantation of AMs without IPE cells.


CONCLUSIONS
The more advanced degree of differentiation of IPE cells grown on AMs indicates that AMs are a better substrate to culture IPE cells than plastic plates. This was supported by the greater protection of photoreceptors of RCS rats when IPE cell sheets cultured on AMs were transplanted in the subretinal space.
Ohno-Matsui
Kyoko
K
Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, Tokyo, Japan. k.ohno.oph@tmd.ac.jp
Mori
Keisuke
K
Ichinose
Shizuko
S
Sato
Tetsuji
T
Wang
Jiying
J
Shimada
Noriaki
N
Kojima
Ariko
A
Mochizuki
Manabu
M
Morita
Ikuo
I
D000650:Amnion / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000295:instrumentation / Q000592:standards; D005136:Eye Proteins / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D007498:Iris / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D009414:Nerve Growth Factors / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000473:pathology; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation / Q000648:ultrastructure; D010969:Plastics; D051381:Rats; D020318:Rats, Long-Evans; D011922:Rats, Mutant Strains; D012160:Retina / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D012164:Retinal Diseases / Q000473:pathology / Q000601:surgery; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D006133:Growth Substances; D009414:Nerve Growth Factors; D010969:Plastics; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor