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Hiroyuki Kamao, Satoshi Okamoto, Michiko Mandai, Yasuhiko Hirami, Junichi Kiryu, Masayo Takahashi; Preparation of iPS-derived RPE Sheets for Transplantation Therapy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4024.
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To prepare a retinal pigment epithelium (RPE) sheet suitable for transplantation therapy.
RPEs derived from human induced pluripotent stem cells were seeded onto type-Icollagen gel on the insert of TranswellTM. After RPEs became confluent, the concentration of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) was measured by ELISA. Then collagenase was added to the collagen gel to release the RPE sheet from the insert. The RPE sheet was then cryosectioned and examined by immunohistochemistry.
It took approximately 4 weeks for the cells to become confluent after they were seeded at 5×105 in the insert. VEGF was mainly secreted into the basal medium, whereas PEDF was mainly secreted into the apical medium. After the addition of collagenase, RPE was separated from the insert as a sheet. An RPE sheet exhibited as monolayer and the junction complex and basal membrane proteins were identified by immunofluorescence labeling as ZO-1, laminin and Collagen-IV. We could follow the same procedure to prepare monkey iPS-derived RPE sheets.
We have established a method to produce RPE sheet that resembles native RPE sheet in morphology, physiology, polarity, and a pattern of protein expressions. Now we similarly produced RPE sheets from monkey iPS cell lines and we plan autologous and allologous transplantations of RPE sheets to test the host immune-reactions.
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