In:
Ophthalmic Research, S. Karger AG, Vol. 43, No. 2 ( 2010), p. 79-91
Abstract:
〈 i 〉 Objective: 〈 /i 〉 It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). 〈 i 〉 pax6 〈 /i 〉 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the 〈 i 〉 pax6 〈 /i 〉 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the 〈 i 〉 pax6 〈 /i 〉 gene. 〈 i 〉 Methods: 〈 /i 〉 Undifferentiated mouse ESCs were transfected with 〈 i 〉 pax6 〈 /i 〉 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of 〈 i 〉 pax6- 〈 /i 〉 transfected cells. We expanded the cloned 〈 i 〉 pax6- 〈 /i 〉 transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. 〈 i 〉 Results: 〈 /i 〉 We obtained clonally expanding 〈 i 〉 pax6 〈 /i 〉 -transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing βIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned 〈 i 〉 pax6- 〈 /i 〉 transfected cells. High KCl stimulation increased free Ca influx on Ca 〈 sup 〉 2+ 〈 /sup 〉 imaging. 〈 i 〉 Conclusions: 〈 /i 〉 ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the 〈 i 〉 pax6 〈 /i 〉 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.
Type of Medium:
Online Resource
ISSN:
0030-3747
,
1423-0259
Language:
English
Publisher:
S. Karger AG
Publication Date:
2010
detail.hit.zdb_id:
1483177-6
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