Direct reprogramming of rat neural precursor cells and fibroblasts into pluripotent stem cells.
BACKGROUND
Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes.


RESULTS
iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency.


CONCLUSIONS
Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease.
Chang
Mi-Yoon
MY
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States of America.
Kim
Dohoon
D
Kim
Chun-Hyung
CH
Kang
Hoon-Chul
HC
Yang
Eungi
E
Moon
Jung-Il
JI
Ko
Sanghyeok
S
Park
Junpil
J
Park
Kyung-Soon
KS
Lee
Kyung-Ah
KA
Hwang
Dong-Youn
DY
Chung
Young
Y
Lanza
Robert
R
Kim
Kwang-Soo
KS
D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology*; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D009474:Neurons / Q000166:cytology*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012190:Retroviridae / Q000235:genetics; D013301:Streptolysins / Q000494:pharmacology
D001426:Bacterial Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D016271:Proto-Oncogene Proteins c-myc; D013301:Streptolysins; C025964:streptolysin O