Expression of leukemia inhibitory factor and its receptors is increased during differentiation of human embryonic stem cells  Lusine Aghajanova, M.D., Ph.D., Heli Skottman, Ph.D., Anne-Marie Strömberg, José Inzunza, Ph.D., Riitta Lahesmaa, M.D., Ph.D., Outi Hovatta, M.D., Ph.D.  Fertility and Sterility  Volume 86, Issue 4, Pages 1193-1209 (October 2006) DOI: 10.1016/j.fertnstert.2005.12.081 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Representative light-microscopic figures of (A) undifferentiated and (B) differentiated hESC colonies of the HS237 line. Scale bar = 100 μm. Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Scanning electron microscopy. Human ESCs in culture: undifferentiated hESCs (globular; A) and differentiated hESCs (B–D). (B) Polygonal cells are forming rosettes. (C) Spindle-like cells. (D) Globular and spindle-like cells. Magnification, ×2,000. Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Microarray results of hESC line HS237. (A) Bar chart representing the number of probe sets, genes with unique Unigene ID, and specific genes for undifferentiated and differentiated hESCs and fibroblasts. (B) Venn diagram of shared and specific genes expressed in undifferentiated and differentiated hESCs and in fibroblasts. The region of overlap between all areas indicates the number of shared genes between compared three cell types. The region of overlap between two areas indicates the number of shared genes between compared two cell types and these genes are not expressed in third cell sample. Nonoverlapping regions indicate genes expressed specifically (not expressed in others) in undifferentiated hESCs, differentiated hESCs, or fibroblasts. (C) Combination Venn diagram of shared and specific genes expressed in undifferentiated and differentiated hESCs. The region of overlap between both areas indicates the number of genes expressed both in undifferentiated and differentiated hESC samples. Nonoverlapping regions indicate genes expressed specifically in undifferentiated or differentiated hESCs. Ellipses represent differentially expressed specific genes, which are not expressed in another cell sample. Rectangles represent genes expressed both in undifferentiated and differentiated hESCs, but with different expression levels. Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Verification of microarray results by means of TaqMan real-time quantitative RT-PCR. The expression patterns of Nanog, OCT-4, UTF1, and DNMT3B (A) and of gp130, LIFR, GATA6, FST, and DUSP6 (B) were analyzed using real-time RT-PCR. Ribonucleic acid was isolated from undifferentiated and differentiated cells of three hESC lines (HS181, HS235, and HS237). All measurements were performed in duplicate in two separate runs. The relative levels of gene expression of target messenger RNA were normalized against GAPDH expression. The average ΔΔCt values of gene expression are presented as fold change (fold change = 2−ΔΔCt) in undifferentiated cells in relation to expression in differentiated cells. Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 TaqMan real-time quantitative RT-PCR analyses of LIFR, gp130, and Nanog in HS293. Human ESCs were cultured without supplementary LIF and with LIF at 5 and 10 ng/mL. All measurements were performed in duplicate in two separate runs. The relative levels of gene expression of target messenger RNA were normalized against GAPDH expression. The average ΔΔCt values of gene expression are presented as fold change (fold change = 2−ΔΔCt) in cells cultured in the presence of LIF as relation to expression in cells cultured without LIF. Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 6 Immunoexpression of LIF, LIF receptors and SOCS-1 in human embryonic stem cells. Undifferentiated hESCs (A–C, G) and differentiated hESCs (D–F, H, I) are shown. LIF expression (A, D), LIFR expression (B, E), gp130 staining (C, F), SOCS-1 immunostaining (G, H), and negative control (I). Magnification, × 400 (A–E and G–I) and ×100 (F). Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 7 Microarray results of components of LIF pathway in hESC line HS237. The expression level of genes up-regulated in undifferentiated (A) or in differentiated (B) cells are represented as fold change in undifferentiated cells in relation to the expression in differentiated cells. Aghajanova. LIF in early differentiated hESCs. Fertil Steril 2006. Fertility and Sterility 2006 86, 1193-1209DOI: (10.1016/j.fertnstert.2005.12.081) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions