title: Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.
abstract: The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.
authors: Renova Life Inc., University of Maryland, TAP program, College Park, MD 20740, USA.
authors: Fei
authors: F
authors: Xue
authors: Yinghong
authors: Y
authors: Ma
authors: Y Eugene
authors: YE
authors: Chen
authors: Jifeng
authors: J
authors: Zhang
authors: Tzu-An
authors: TA
authors: Lin
authors: Chien-Hong
authors: CH
authors: Chen
authors: Wei-Wen
authors: WW
authors: Lin
authors: Marsha
authors: M
authors: Roach
authors: Jyh-Cherng
authors: JC
authors: Ju
authors: Lan
authors: L
authors: Yang
authors: Fuliang
authors: F
authors: Du
authors: Jie
authors: J
authors: Xu
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D018920:Coculture Techniques / Q000379:methods; D004622:Embryo, Mammalian / Q000166:cytology* / Q000196:embryology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005855:Germ Layers / Q000166:cytology / Q000196:embryology; D006801:Humans; D000090062:Kruppel-Like Factor 4; D053605:Leukemia Inhibitory Factor / Q000096:biosynthesis* / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D053605:Leukemia Inhibitory Factor; D011994:Recombinant Proteins; D014157:Transcription Factors