Pluripotin combined with leukemia inhibitory factor greatly promotes the derivation of embryonic stem cell lines from refractory strains.
Most mouse embryonic stem (ES) cells are derived from a 129 or C57BL/6 background, whereas the derivation efficiency of ES cells is extremely low on certain refractory types of background for which ES cells are highly desired. Here we report an optimized, highly efficient protocol by combining pluripotin, a small molecule, and leukemia inhibitory factor (LIF) for the derivation of mouse ES cells. With this method, we successfully isolated ES cell lines from five strains of mice, with an efficiency of 57% for NOD-scid, 63% for SCID beige, 80% for CD-1, and 100% for two F1 strains from C57BL/6xCD-1. By tracking the Oct4-positive cells in the Oct4-green fluorescent protein embryos in the process of ES cell isolation, we found that pluripotin combined with LIF improved the efficiency of ES cell isolation by selectively maintaining the Oct4-positive cells in the outgrowth. To our knowledge, this is the first report of ES cells being efficiently derived from immunodeficient mice on refractory backgrounds (NOD-scid on a NOD background and SCID beige on a BALB/c background).
Yang
Weifeng
W
Laboratory of Stem Cell and Generative Biology, College of Life Sciences, Peking University, Beijing, China.
Wei
Wei
W
Shi
Cheng
C
Zhu
Jinliang
J
Ying
Wenqin
W
Shen
Yan
Y
Ye
Xin
X
Fang
Lingling
L
Duo
Shuguang
S
Che
Jie
J
Shen
Huan
H
Ding
Sheng
S
Deng
Hongkui
H
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D007150:Immunohistochemistry; D007621:Karyotyping; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008822:Mice, Transgenic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D053605:Leukemia Inhibitory Factor