Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells.
We previously reported that transfection of Pax6 gene which regulated early events in eye development into mouse ES cells brought about their differentiation into retinal progenitors. Here, we attempted to establish cloned retinal progenitors which had ability to further differentiate into photoreceptor like cells by transfecting mouse induced pluripotent stem (iPS) cells with Pax6 gene. Undifferentiated iPS cells were transfected with Pax6 cDNA, followed by selection with G418. After limiting dilution culture, we selected cloned Pax6-transfected cells, which simultaneously expressed mRNAs of Nestin, Musashi1, Six3 and Chx10 for further characterization. We obtained totally 8 clonally expanding Pax6-transfected cells. They started to express mRNAs of Brn3b, Cone-rod homeobox (Crx), pkc, CD73, rhodopsin and the γ-subunit of rod cGMP phosphodiesterase (PDEγ). Flow cytometric analysis revealed that almost half of the cells were CD73+, a marker of photoreceptor precursors. Western blotting confirmed cytoplasmic protein expression of rhodopsin. High KCl stimulation increased free Ca influx into the cells on Ca(2+) imaging. iPS cells transfected with Pax6 gene, followed by subsequent limiting dilution culture became retinal progenitors including photoreceptor like cells. The cloned cell lines may be useful for analyzing differentiation requirement of retinal progenitors.
Department of Immunology, St. Marianna University School of Medicine, Kawasaki, Japan. n3suzuki@marianna-u.ac.jp
Noboru
N
Suzuki
Jun
J
Shimizu
Kenji
K
Takai
Nagisa
N
Arimitsu
Yuji
Y
Ueda
Erika
E
Takada
Chieko
C
Hirotsu
Tomoko
T
Suzuki
Naruyoshi
N
Fujiwara
Mamoru
M
Tadokoro
D000818:Animals; D002118:Calcium / Q000032:analysis; D020013:Calcium Signaling; D002460:Cell Line; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D012160:Retina / Q000166:cytology* / Q000378:metabolism*; D014162:Transfection
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D002118:Calcium