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. Author manuscript; available in PMC: 2011 Aug 26.
Published in final edited form as: Cell Stem Cell. 2010 Jun 4;6(6):535–546. doi: 10.1016/j.stem.2010.05.003

Figure 3. The hLR5 state depends on ectopic pluripotency factors but is poised for re-activation of endogenous pluripotency genes.

Figure 3

(A) Ectopic factor dependence of hLR5 cells. Upon doxycycline withdrawal, hLR5 colony morphology is lost and cells adopt a fibroblast-like appearance. Days of differentiation are indicated

(B) Quantitative RT-PCR analysis of the expression of reprogramming factors used for the derivation and maintenance of hLR5 cells. Left panel: expression of endogenous genes. Right panel: expression of the Doxycyclin-inducible ectopic reprogramming factors. Human ES strains (H9, HUES3, HUES14) and human iPS strains (hiPS1, hiPS2) were used as controls. Color coding of the genes is indicated (n-3, SD).

(C) ChIP-qPCR analysis of the presence of Histone 3 lysine 4 (H3K4, green bars) marks and Histone 3 Lysine 27 (H3K27, red bars) marks at the promoter regions of the pluripotency genes SOX2, DNMT3b and SALL4 as indicated in hLR5 cells (n=3, SD).

(D) ChIP-qPCR analysis of the presence of Histone 3 lysine 4 (H3K4, green bars) marks and Histone 3 Lysine27 (H3K27, red bars) marks at the promoter regions of the pluripotency genes OCT4, NANOG and REX1 as indicated in hLR5 cells(n=3, SD).

(E) DNA methylation analysis of two CpG islands in the OCT4 promoter as indicated in the schematic of the OCT4 promoter region. Open circles indicate unmethylated and filled circles indicate methylated CpG dinucleotides. Shown are representative sequenced clones from BJ fibroblasts, human iPS cells and two independent clonal hLR5 cell lines. The percentage of CpG methylation at each CpG island in the respective cell lines is indicated. TSS: Transcription start site

(F) Schematic representation of the generation of hLR5 cells in the absence (Top panel, I.) or presence (Bottom panel, II.) of NANOG. While in the absence of NANOG expression traditional hiPS cell can be derived, no hLR5-like colonies form. Addition of ectopic NANOG results in the formation of hLR5 colonies.