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Linking the p53 tumour suppressor pathway to somatic cell reprogramming.

Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, Wahl GM, Izpisúa Belmonte JC - Nature (2009)

Bottom Line: We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells.Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells.These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.

ABSTRACT
Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

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Generation and characterization of 2F-p53KD-iPS cells by p53 downregulation(a) Morphology and GFP fluorescence of 2F-p53KD-iPS cell lines. GFP expression is silenced in clone #6. (b) Alkaline phosphatase staining of 2F-p53KD-iPS cell lines. DAPI was used to visualize cell nuclei. (c) Protein levels of Nanog, Oct4, Sox2, Klf4, c-Myc, p53 in 2F-p53KD-iPS cell lines are shown. α-Tubulin was used as loading control. (d) Embryoid bodies (EBs) of 2F-p53KD-iPS cell clones on day 6 of differentiation. EBs were transferred to gelatinized dishes on day 3 to 5 for further differentiation. On day 14, EBs were subjected to immunofluorescence for α-fetoprotein (AFP)/Foxa2 (endoderm), α-sarcomeric actin/GATA4 (mesoderm) and Tuj1/GFAP (ectoderm). (e) Immunofluorescence of teratoma from 2F-p53KD-iPS cells by antibodies against AFP/Foxa2 (endoderm), α-sarcomeric actinin/Chondroitin (mesoderm), Tuj1/GFAP (ectoderm) showed spontaneous differentiation into all three germ layers. (f) Adult chimeric mice obtained from 2F-p53KD iPS lines (#1 and #6) and non-chimeric mouse in C57BL/6J host blastocysts. (g) As of the date of submission, the mating of offspring from clone #6 chimera to a C57BL/6J female generated 1 agouti pup (blue arrow), that together with PCR analysis (not shown) indicate germ line transmission of the 2F-iPS genome.
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Figure 3: Generation and characterization of 2F-p53KD-iPS cells by p53 downregulation(a) Morphology and GFP fluorescence of 2F-p53KD-iPS cell lines. GFP expression is silenced in clone #6. (b) Alkaline phosphatase staining of 2F-p53KD-iPS cell lines. DAPI was used to visualize cell nuclei. (c) Protein levels of Nanog, Oct4, Sox2, Klf4, c-Myc, p53 in 2F-p53KD-iPS cell lines are shown. α-Tubulin was used as loading control. (d) Embryoid bodies (EBs) of 2F-p53KD-iPS cell clones on day 6 of differentiation. EBs were transferred to gelatinized dishes on day 3 to 5 for further differentiation. On day 14, EBs were subjected to immunofluorescence for α-fetoprotein (AFP)/Foxa2 (endoderm), α-sarcomeric actin/GATA4 (mesoderm) and Tuj1/GFAP (ectoderm). (e) Immunofluorescence of teratoma from 2F-p53KD-iPS cells by antibodies against AFP/Foxa2 (endoderm), α-sarcomeric actinin/Chondroitin (mesoderm), Tuj1/GFAP (ectoderm) showed spontaneous differentiation into all three germ layers. (f) Adult chimeric mice obtained from 2F-p53KD iPS lines (#1 and #6) and non-chimeric mouse in C57BL/6J host blastocysts. (g) As of the date of submission, the mating of offspring from clone #6 chimera to a C57BL/6J female generated 1 agouti pup (blue arrow), that together with PCR analysis (not shown) indicate germ line transmission of the 2F-iPS genome.

Mentions: Since Klf4 has been reported to have oncogenic properties when overexpressed22, and we showed that it alone can activate p53, we investigated whether cells with reduced p53 expression could be converted into iPS cells using only two factors, Oct4, and Sox2. We tested this hypothesis by transducing MEFs with a lentivirus expressing p53 shRNA plus retroviruses encoding Oct4 and Sox2 (hereafter designated as 2F-p53KD-iPS cells; Supplementary Fig. 13). Cells that developed into colonies exhibiting ES cell-like morphology were obtained by week four post-infection. Of six colonies selected for analysis, four grew using standard mouse ES cell culturing methods (Fig. 3a), and all of them were alkaline phosphatase positive (Fig. 3b) and expressed genes and cell surface markers characteristic of mouse ES cells including the pluripotency marker Nanog (Fig. 3c; Supplementary Fig. 14). 2F-p53KD-iPS cells and mouse ES cell lines exhibited indistinguishable gene expression patterns when maintained under similar conditions. Bisulfite sequencing of the Oct4 and Nanog promoters revealed nearly complete demethylation in 2F-p53KD-iPS cells when compared to MEFs (Supplementary Fig. 14). Consistent with this, we observed expression of the pluripotency-associated transcription factors Oct4 and Sox2 from the endogenous loci in 2F-p53KD-iPS cells, at levels that were comparable to those of ES cells (Supplementary Fig. 15). Also, like ES cells, the majority (70–80%) of cells were in S-phase (Supplementary Fig. 16). We tested the pluripotency of three 2F-p53KD-iPS clones in assays of embryoid body formation in vitro and/or teratoma induction in vivo.. The tested cell lines differentiated into the three germ layer derivatives, as shown by immunostaining and mRNA expression in vitro (Fig. 3d; Supplementary Fig. 17). Furthermore, these cells differentiated with high efficiency into beating cardiomyocytes (Supplementary Fig. 18 and movies 1–3). Upon injection into immunocompromised mice, two independent 2F-p53KD-iPS lines generated complex intratesticular and subcutaneous teratomas containing structures and tissues representative of the three embryonic germ layers (Fig. 3e). Microarray analyses demonstrate that gene expression patterns of these clones are similar to mouse ES cells (Supplementary Fig. 19). We also tested whether 2F-p53KD-iPS cells contribute to the formation of chimeric mice when injected into mouse blastocysts. One line (clone#6) contributed almost 100% to chimera formation, and the other line (clone#1) contributed 30–50%, as judged by coat color (Fig. 3f; Supplementary Fig. 20). We finally used the highest contribution chimera to test for germline competence by crossing it with wild-type C57BL6 mice. Importantly, the offspring of such crosses included agouti pups (Fig. 3g), indicating germline transmission of the 2F-iPS genome. Taken together, these results demonstrate that MEFs can be reprogrammed to pluripotency by the forced expression of only two factors, Oct4 and Sox2, when p53 levels are reduced.


Linking the p53 tumour suppressor pathway to somatic cell reprogramming.

Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, Wahl GM, Izpisúa Belmonte JC - Nature (2009)

Generation and characterization of 2F-p53KD-iPS cells by p53 downregulation(a) Morphology and GFP fluorescence of 2F-p53KD-iPS cell lines. GFP expression is silenced in clone #6. (b) Alkaline phosphatase staining of 2F-p53KD-iPS cell lines. DAPI was used to visualize cell nuclei. (c) Protein levels of Nanog, Oct4, Sox2, Klf4, c-Myc, p53 in 2F-p53KD-iPS cell lines are shown. α-Tubulin was used as loading control. (d) Embryoid bodies (EBs) of 2F-p53KD-iPS cell clones on day 6 of differentiation. EBs were transferred to gelatinized dishes on day 3 to 5 for further differentiation. On day 14, EBs were subjected to immunofluorescence for α-fetoprotein (AFP)/Foxa2 (endoderm), α-sarcomeric actin/GATA4 (mesoderm) and Tuj1/GFAP (ectoderm). (e) Immunofluorescence of teratoma from 2F-p53KD-iPS cells by antibodies against AFP/Foxa2 (endoderm), α-sarcomeric actinin/Chondroitin (mesoderm), Tuj1/GFAP (ectoderm) showed spontaneous differentiation into all three germ layers. (f) Adult chimeric mice obtained from 2F-p53KD iPS lines (#1 and #6) and non-chimeric mouse in C57BL/6J host blastocysts. (g) As of the date of submission, the mating of offspring from clone #6 chimera to a C57BL/6J female generated 1 agouti pup (blue arrow), that together with PCR analysis (not shown) indicate germ line transmission of the 2F-iPS genome.
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Figure 3: Generation and characterization of 2F-p53KD-iPS cells by p53 downregulation(a) Morphology and GFP fluorescence of 2F-p53KD-iPS cell lines. GFP expression is silenced in clone #6. (b) Alkaline phosphatase staining of 2F-p53KD-iPS cell lines. DAPI was used to visualize cell nuclei. (c) Protein levels of Nanog, Oct4, Sox2, Klf4, c-Myc, p53 in 2F-p53KD-iPS cell lines are shown. α-Tubulin was used as loading control. (d) Embryoid bodies (EBs) of 2F-p53KD-iPS cell clones on day 6 of differentiation. EBs were transferred to gelatinized dishes on day 3 to 5 for further differentiation. On day 14, EBs were subjected to immunofluorescence for α-fetoprotein (AFP)/Foxa2 (endoderm), α-sarcomeric actin/GATA4 (mesoderm) and Tuj1/GFAP (ectoderm). (e) Immunofluorescence of teratoma from 2F-p53KD-iPS cells by antibodies against AFP/Foxa2 (endoderm), α-sarcomeric actinin/Chondroitin (mesoderm), Tuj1/GFAP (ectoderm) showed spontaneous differentiation into all three germ layers. (f) Adult chimeric mice obtained from 2F-p53KD iPS lines (#1 and #6) and non-chimeric mouse in C57BL/6J host blastocysts. (g) As of the date of submission, the mating of offspring from clone #6 chimera to a C57BL/6J female generated 1 agouti pup (blue arrow), that together with PCR analysis (not shown) indicate germ line transmission of the 2F-iPS genome.
Mentions: Since Klf4 has been reported to have oncogenic properties when overexpressed22, and we showed that it alone can activate p53, we investigated whether cells with reduced p53 expression could be converted into iPS cells using only two factors, Oct4, and Sox2. We tested this hypothesis by transducing MEFs with a lentivirus expressing p53 shRNA plus retroviruses encoding Oct4 and Sox2 (hereafter designated as 2F-p53KD-iPS cells; Supplementary Fig. 13). Cells that developed into colonies exhibiting ES cell-like morphology were obtained by week four post-infection. Of six colonies selected for analysis, four grew using standard mouse ES cell culturing methods (Fig. 3a), and all of them were alkaline phosphatase positive (Fig. 3b) and expressed genes and cell surface markers characteristic of mouse ES cells including the pluripotency marker Nanog (Fig. 3c; Supplementary Fig. 14). 2F-p53KD-iPS cells and mouse ES cell lines exhibited indistinguishable gene expression patterns when maintained under similar conditions. Bisulfite sequencing of the Oct4 and Nanog promoters revealed nearly complete demethylation in 2F-p53KD-iPS cells when compared to MEFs (Supplementary Fig. 14). Consistent with this, we observed expression of the pluripotency-associated transcription factors Oct4 and Sox2 from the endogenous loci in 2F-p53KD-iPS cells, at levels that were comparable to those of ES cells (Supplementary Fig. 15). Also, like ES cells, the majority (70–80%) of cells were in S-phase (Supplementary Fig. 16). We tested the pluripotency of three 2F-p53KD-iPS clones in assays of embryoid body formation in vitro and/or teratoma induction in vivo.. The tested cell lines differentiated into the three germ layer derivatives, as shown by immunostaining and mRNA expression in vitro (Fig. 3d; Supplementary Fig. 17). Furthermore, these cells differentiated with high efficiency into beating cardiomyocytes (Supplementary Fig. 18 and movies 1–3). Upon injection into immunocompromised mice, two independent 2F-p53KD-iPS lines generated complex intratesticular and subcutaneous teratomas containing structures and tissues representative of the three embryonic germ layers (Fig. 3e). Microarray analyses demonstrate that gene expression patterns of these clones are similar to mouse ES cells (Supplementary Fig. 19). We also tested whether 2F-p53KD-iPS cells contribute to the formation of chimeric mice when injected into mouse blastocysts. One line (clone#6) contributed almost 100% to chimera formation, and the other line (clone#1) contributed 30–50%, as judged by coat color (Fig. 3f; Supplementary Fig. 20). We finally used the highest contribution chimera to test for germline competence by crossing it with wild-type C57BL6 mice. Importantly, the offspring of such crosses included agouti pups (Fig. 3g), indicating germline transmission of the 2F-iPS genome. Taken together, these results demonstrate that MEFs can be reprogrammed to pluripotency by the forced expression of only two factors, Oct4 and Sox2, when p53 levels are reduced.

Bottom Line: We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells.Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells.These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.

ABSTRACT
Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

Show MeSH
Related in: MedlinePlus