1. Requirement of leukemia inhibitory factor for establishing and maintaining embryonic stem cells in mice 
Jae Hee Lee, B.Sc., Eun Ju Lee, Ph.D., Chae Hyun Lee, B.Sc., Jun Hong Park, Ph.D., Jae Yong Han, Ph.D., Jeong Mook Lim, D.V.M., Ph.D. 
Fertility and Sterility 
Volume 92, Issue 3, Pages (September 2009)
DOI: /j.fertnstert
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Formation of colonies derived from inner cell mass (ICM) cells of blastocysts cultured in leukemia inhibitory factor (LIF)–free or 2,000 U/mL LIF–containing medium and mRNA expression of the genes regulating stemness. Messenger RNA expression of the genes regulating self-renewal (Oct4 and Nanog) and LIF signaling (gp130 and Stat3) in ICM colonies cultured for 4 days (primary culture). The expression level was quantified by real-time polymerase chain reaction, and the level was normalized with that of β-actin mRNA. The expression of Oct4, Nanog, and gp130 genes was increased at no LIF addition, but no statistical significance was detected (P>.3457). A significant decrease in Stat3 expression (P<.001) was detected at no LIF addition.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Proliferation and morphology of mouse embryonic stem cells (ESCs) on fibroblast feeder layer. Dulbecco minimal essential medium supplemented with fetal bovine serum was used as a base medium, to which 0 or 1,000 U/mL leukemia inhibitory factor (LIF) was added. (A) Morphology of E14 or R1 ESCs was monitored at the tenth subculture. Clonal colony formation was detected in all treatments, and no prominent difference in the morphology of colonies was detected. Scale bar = 50 μm. (B) Size of colonies positively stained with alkaline phosphatase (AP) and the ratio of the area positively stained with AP to the area with no reactivity to AP (called area fraction). Each value derived from 0 or 1,000 U/mL LIF was compared by Image J software (National Institutes of Health, Bethesda, MD) on day 3 of culture. There was no significant change in area fraction (P=.3563) or colony size (P=.1012) between no LIF and 1,000 U/mL LIF additions.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Messenger RNA expression of the genes regulating stemness of mouse embryonic stem cells (ESCs) cultured in leukemia inhibitory factor (LIF)–free or 1,000 U/mL LIF–containing medium. Dulbecco minimal essential medium supplemented with fetal bovine serum was used as a base medium for culturing E14 or R1 ESCs. (A) Meseenger RNA expression of the genes mediating LIF function (LIF-JAK-STAT pathway; gp130, Stat3, and c-myc) in ESCs collected at the tenth subpassage was quantified by real-time polymerase chain reaction, and the expression levels were normalized with that of β-actin mRNA. (B) Messenger RNA expression of the genes regulating stem cell self-renewal (Oct4 and Nanog) and cell growth/regulation (Tert, Dmnt3b, and Spin). (C) Messenger RNA expression of the genes regulating ectodermal (S-100), mesodermal (SM-actin), or endodermal (α-fetoprotein) differentiation. Overall, changes in gene expression were detected at different LIF levels, but no statistical (P>.1703) difference was found in all of the comparisons.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Embryoid body (EB) formation of mouse embryonic stem cells (ESCs) maintained in the medium supplemented with 0 or 1,000 U/mL leukemia inhibitory factor (LIF). (A) E14 or R1 ESCs subpassaged five or ten times were provided for the EB formation, and a hanging drop method of LIF- and feeder-free culture system was used. The morphology of EBs formed was observed on day 4 of induction. (B) Formation efficiency of EBs derived from ESCs maintained in LIF-free or 1,000 U/mL LIF–containing medium, which was calculated by the percentage of the number of EBs formed to total number of hanging drops containing ESCs. There were no significant differences (P>.5121) in the morphology and the EB formation efficiency.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions