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Hirofumi Kinoshita, Kiyoshi Suzuma, Jun Kaneko, Michiko Mandai, Takashi Kitaoka, Masayo Takahashi; Generation of ciliary epithelium from mouse ES and iPS cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1386.
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Recently, Eiraku et al reported a protocol for self-organizing optic-cup morphogenesis in three-dimensional culture. With this procedure, we could differentiate mouse embryonic stem (ES) cells or mouse induced pluripotent stem (iPS) cells into neural retina, retinal pigment epithelium (RPE), as well as tissue derived from neural ectoderm in the three-dimensional structure. Here, we report a possibility that ciliary epithelium, which is essential for production of aqueous humor, could be differentiated from mouse ES/iPS cells.
Mouse ES/iPS cells (Rx knock-in GFP ES line mESRx+; Nrlp-eGFP transgenic iPS line) were maintained in maintenance culture as it might lead to undesirable differentiation of cells. Cells were differentiated by using modified Eiraku’s differentiation protocol, induced optic-cup-like structures were investigated morphologically, or immunohistochemically. E16.5 and P5 of C57BL/6J mice were evaluated as control.
We could show autonomous formation of the optic-cup-like structure from a three-dimensional culture of mouse ES/iPS cell aggregates by using modified Eiraku's differentiation protocol. To evaluate whether there is a development of ciliary epithelium in this optic-cup-like structure, we identified immunohistochemistry for ciliary marker. Prospective ciliary epitheliums, bilayer of pigmented and non-pigmented epithelium (PE and NPE), were seen in peripheral region of inner and outer epithelium in E16.5 mouse eye. Aquaporin1-positive staining was seen in the NPE. Connexin43-positive staining was seen in the ciliary epitheliums (PE and NPE), RPE and gap junction, which is structure between PE and NPE. In P5 mouse eye, neural retina and ciliary epithelium became morphologically distinguishable more clearly. Aquaporin1-positive staining was seen in the NPE, especially in the anterior pars plicata. Aquaporin4-positive staining was seen in the NPE, connexin43-positive staining was seen in the ciliary epitheliums (PE and NPE), RPE and gap junction. Differentiated structures derived from mouse ES cells were Rx-GFP positive. In immunohistochemistry of aggregates derived from mouse iPS cells, expression of aquaporin1, connexin43 were positive in ciliary epithelium like structures.
We could differentiate the ciliary epithelium like structures from mouse ES/iPS cells. These complexes showed similarity of morphology and immunostaining of ciliary epitheliums in vivo.
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