Transfection with pax6 gene of mouse embryonic stem cells and subsequent cell cloning induced retinal neuron progenitors, including retinal ganglion cell-like cells, in vitro.
OBJECTIVE
It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 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 pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene.


METHODS
Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-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.


RESULTS
We obtained clonally expanding pax6-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 betaIII 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 pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging.


CONCLUSIONS
ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 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.
Kayama
Maki
M
Department of Ophthalmology, St. Marianna University School of Medicine, Kawasaki, Japan.
Kurokawa
Manae S
MS
Ueda
Yuji
Y
Ueno
Hiroki
H
Kumagai
Yuta
Y
Chiba
Shummei
S
Takada
Erika
E
Ueno
Satoki
S
Tadokoro
Mamoru
M
Suzuki
Noboru
N
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D018274:Electroporation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D005353:Fibronectins / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D012165:Retinal Ganglion Cells / Q000166:cytology*; D055351:Retinal Neurons / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D013370:Subrenal Capsule Assay; D014162:Transfection
D015415:Biomarkers; D005136:Eye Proteins; D005353:Fibronectins; D018398:Homeodomain Proteins; D007381:Intermediate Filament Proteins; C104786:Msi1h protein, mouse; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D016601:RNA-Binding Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D012097:Repressor Proteins; D002118:Calcium