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First published online February 24, 2006
doi: 10.1242/10.1242/dev.02286

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Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features

Department of Genetics, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel

View larger version (29K): [in a new window]   Fig. 1. Establishment of NANOG overexpressing HESC clones and examination of their growth. (A, part I) Establishment of NANOG overexpressing HESC clones was verified following selection with puromycin by RT-PCR. Amplification was performed using specific primers for the NANOG transgene. GAPDH was used as a positive control. (II) The level of NANOG overexpression was analyzed by real-time RT-PCR to examine the total NANOG mRNA level in the cells. The three clones examined showed significant elevation of the mRNA level (*P<0.05). (III) Western blot analysis verified that the elevation in the mRNA levels corresponded to an elevation at the protein level. TUBULIN was used as the loading control. (B) Cells (2x104 per cm2) were seeded on plates coated with gelatin and grown either in the presence (top) or absence (bottom) of conditioned media. To analyze the growth rate of the cells, they were fixed in 0.5% glutardialdehyde after the indicated number of days. Cell staining was performed using Methylene Blue, color was extracted with 0.1 M HCl and absorbance (650 nm) was used as an indication of relative cell number. Error bars represent s.e.m.

View larger version (56K): [in a new window]   Fig. 2. NANOG overexpressing cells can be serially passaged in the absence of feeders. (A) Wild-type and NANOG overexpressing cells were seeded on laminin-coated plates at a density of 2x104 cells per cm2 and passaged in the absence of conditioned media. Photos were taken of the third passage after withdrawal of conditioned media. Whereas wild-type cells stopped proliferating and showed differentiated morphology, NANOG overexpressing cells continued to proliferate for more than 15 passages and formed colonies with ES cell morphology. (B) OCT4 expression was examined on the 15th passage and was still positive in most of the cells. (C) TRA-1-60 staining performed 20 passages after CM withdrawal showed that the majority of the cells (more than 90%) remained undifferentiated. (I) MEF cells only, (II) wild-type cells grown on feeders, (III) clone grown on feeders, (IV) clone grown for 20 passages without feeders or CM. (D) After 15 passages in the absence of CM, the cells still formed teratomas composed of multiple cell types following injection into immunodeficient mice.

View larger version (36K): [in a new window]   Fig. 3. Formation of feeder-free colonies is dependent on NANOG expression. (A) Five-hundred cells per cm2 from H9 and H13 cell lines were seeded in 12-well plates coated with gelatin and FCS. After 8 days, the cells were fixed and stained for alkaline phosphatase (AP) activity. Positively stained colonies were counted. The results shown for H13 clones represent the average of three independent clones. Scale bars represent s.e.m. (*P<0.05). (B) Representative photos of each clone. AP staining of undifferentiated cells appears as red staining. (C) The experiment described in A was repeated after establishment of revertant clones from which the transgene was removed using the CRE-loxP system. Shown are results of the parental cells and the average of three individual CRE clones derived from it. Error bars represent s.e.m. (*P<0.05). (D) Representative photos of the results depicted in C.

View larger version (27K): [in a new window]   Fig. 4. NANOG is upregulated upon early differentiation. (A) The expression pattern of NANOG during differentiation was examined by real-time RT-PCR. Four stages of in vitro differentiation were examined: ES, EB2d, EB10d and EB30d. Shown is the abundance of NANOG transcript normalized to GAPDH levels per sample. Error bars represent s.e.m. (B) The expression pattern revealed by real-time RT-PCR was verified by semi-quantitative RT-PCR under non-saturating conditions. The temporal expression pattern of NANOG was compared with that of OCT4, which, unlike NANOG, does not show elevation of expression during differentiation. NTC (no templates control) was used as a negative control. GAPDH was used as positive control.

View larger version (31K): [in a new window]   Fig. 5. NANOG overexpressing cells adopt markers of primitive ectoderm cells. (A) The ICM (inner cell mass) and epiblast (primitive ectoderm) can be distinguished by the differential expression of a subset of marker genes. ICM has a high expression of Rex1 and Gbx2, and low expression of Fgf5. Epiblast has low expression of Rex1 and Gbx2, and upregulation of Fgf5. (B) Real-time RT-PCR analysis was performed on markers that distinguish between ICM and primitive ectoderm cells. Expression was examined on wild-type cells and on NANOG overexpressing clones (shown as the average of three separate clones). Although GAPDH and OCT4 did not show a significant change, REX1 and GBX2 were significantly downregulated, and FGF5 was significantly upregulated following NANOG overexpression (*P<0.05). (C) Semi-quantitative RT-PCR analysis was executed on the markers described in B. Expression was examined in wild-type cells, NANOG overexpressing clones and revertant clones from which the transgene was excised. Shown are representative pictures of the examined clones. (D) RT-PCR analysis was performed on Nanog overexpressing MESCs. A comparison was performed between two separate MESC lines (E14Tg2a and RF8) and clones overexpressing Nanog derived from them.

View larger version (28K): [in a new window]   Fig. 6. Transcriptome analysis of NANOG overexpressing cells. DNA microarray analysis was performed on RNA extracted from NANOG overexpressing cells, wild-type ES cells, EB2d, EB10d and EB30d. Analysis was performed using the U133A chip from Affymetrix, each experiment was performed in triplicate and each DNA microarray was normalized to an average value of 100. (A) Dendogram analysis reveals that upon overexpression of NANOG, the similarity of the cells to cells of EB2d increases. (B) Differentially expressed genes were divided into four groups: (I) genes upregulated in NANOG overexpressing cells and in EB2d compared with ES cells; (II) genes upregulated in NANOG overexpressing cells compared with both ES cells and EB2d; (III) genes downregulated in both NANOG overexpressing cells and in EB2d compared with ES cells; and (IV) genes downregulated in NANOG overexpressing cells compared with both ES cells and EB2d. (C) Shown are genes upregulated in NANOG overexpressing cells in comparison with wild-type ES cells. All the genes shown are at least fourfold upregulated in NANOG overexpressing cells. (D) Shown are genes downregulated in NANOG overexpressing cells in comparison with wild-type ES cells. All the genes shown are downregulated by at least 25-fold in NANOG overexpressing cells.

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