Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

Abstract

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1-8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.

Figure 1

Removal of lamin A/C from cells incubated in ES cell or Xenopus egg extracts. (A) Western blot of Nanog, Oct4, Lamin A/C and Lamin B in MEF and 46C ES cells. (B, C) MEF cells were permeabilized and incubated in 46C ES cell extract or Xenopus egg extract for 3 hours in plus or minus apyrase. Cells were then stained for lamin A/C (FITC, green), lamin B2 (Cy-3, red) or DNA (DAPI, blue). Quantification in (C) was performed by visual inspection. Scale bar, 5μm.

Figure 2

Activation of pluripotency genes in permeabilized 293T cells exposed to mouse ES cell extracts. (A, B) Quantitative RT-PCR analysis of indicated genes in EC or ES cells relative to differentiated 293T or 3T3 cells. In A, human specific-primers were used, whilst in B, primers recognising both human and mouse genes were used. NA - no amplification. (C-F) Quantitative RT-PCR analysis using human-specific primers of gene expression in 293T cells treated for 0 to 8 hours with: (C, F) mouse J1 ES cell extract, (D) mouse 46C ES cell extract and (E) mouse 3T3 cell extract. In (F), unpermeabilised 293 cells were used.

Figure 3

Absence of reprogramming activity in Xenopus egg or oocyte extracts. Quantitative RT-PCR analysis of expression of indicated genes in 293T cells treated for 0 to 8 hours with: (A) Xenopus egg extract, (B) Xenopus oocyte extract or (C) mixes of ES cell and Xenopus oocyte extracts for 6 hours.

Figure 4

Efficient induction of pluripotency genes depends on transcription and translation. (A, B) Chromatin immunoprecipitation, using an RNA polymerase II antibody, on either untreated NCCIT or 293 cells (A) or 293T cells incubated in 46C ES cell extract for 0 to 6 hours (B). Chromatin immunoprecipitates were subjected to PCR using primers specific for GAPDH, Nanog (promoter 1), Oct4 and CRIPTO. W, water only control for PCR reaction. The Nanog promoter (C) Quantitative RT-PCR analysis after incubation of 293T cells for 6 hours in mouse 46C ES extract. The ES extract was optionally supplemented with 100 μM α-amanitin or 1 mg/ml cycloheximide at the start of the incubation. Expression is expressed relative to untreated 293T cells.

Figure 5

46C ES cell extract induces histone modifications at pluripotency gene promoters. Chromatin immunoprecipitation was performed on (A) NCCIT and 293T cells or (B-D) on permeabilized 293T cells exposed to mouse ES cell extract for 0, 2, 4 or 6 hr. Chromatin was precipitated with antibodies to histone H3 modifications and DNA was amplified using primers against GAPDH, Nanog (promoter 2), Oct4 and CRIPTO. W, water only control for PCR reaction. In B-D, input DNA and chromatin precipitated with non-immune IgG

Figure 6

Reprogrammed gene expression increases after return to cell culture. Human 293T cells were permeabilized with SLO and incubated for 6 hours in 46C ES cell extract. Cell membranes were then allowed to reseal for 2 hours, and the cells were returned to tissue culture medium. At the indicated times afterwards, quantitative RT-PCR was performed on selected genes. Expression was normalised to the level of lamin B2 expression and is expressed relative to levels in untreated 293T cells. Error bars show the standard deviation from 3 replicates.