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Gene expression profiling in the reprogramming process

ABSTRACT: It remains unclear how the ectopic expression of defined transcription factors induces dynamic changes in gene expression profiles that establish a pluripotent state during direct cell reprogramming. In the present study, we first identified a temporal gene expression program during the reprogramming process. Promoter analyses then predicted the role of two forkhead box transcription factors, Foxd1 and Foxo1, as mediators of the gene expression program. Knockdown of Foxd1 or Foxo1 reduced the number of induced pluripotent stem cells (iPSCs). The knockout of Foxd1 prevented the downstream transcription program, including the expression of reprogramming marker genes. Interestingly, the expression level of Foxd1 was also transiently increased in a small population of cells in the middle stage of reprogramming. The presence or absence of Foxd1 expression in this stage was correlated with a future cell fate as iPSCs or non-reprogrammed cells. These results suggest that Foxd1 is a mediator and indicator of the successful progression of the gene expression program in cell reprogramming. Mouse embryonic fibroblasts (MEFs) were infected with retroviruses encoding either the four transcription factors (Oct4, Sox2, Klf4 and c-Myc) or GFP (control) at day 0 and sampled on the indicated days. Two replicates each.

ORGANISM(S): Mus musculus

SUBMITTER: Miki Ebisuya

PROVIDER: E-GEOD-38509 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Effect of Foxd1 knockdown in the reprogramming process

Project description:It remains unclear how the ectopic expression of defined transcription factors induces dynamic changes in gene expression profiles that establish a pluripotent state during direct cell reprogramming. In the present study, we first identified a temporal gene expression program during the reprogramming process. Promoter analyses then predicted the role of two forkhead box transcription factors, Foxd1 and Foxo1, as mediators of the gene expression program. Knockdown of Foxd1 or Foxo1 reduced the number of induced pluripotent stem cells (iPSCs). The knockout of Foxd1 prevented the downstream transcription program, including the expression of reprogramming marker genes. Interestingly, the expression level of Foxd1 was also transiently increased in a small population of cells in the middle stage of reprogramming. The presence or absence of Foxd1 expression in this stage was correlated with a future cell fate as iPSCs or non-reprogrammed cells. These results suggest that Foxd1 is a mediator and indicator of the successful progression of the gene expression program in cell reprogramming. Mouse embryonic fibroblasts (MEFs) of Foxd1+/+, Foxd1+/- or Foxd1-/- were infected with retroviruses encoding the three transcription factors (Oct4, Sox2 and Klf4) at day 0 and sampled at day 8.

2014-01-11 | E-GEOD-51179 | biostudies-arrayexpress

Effect of Foxd1 knockdown in the reprogramming process

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Gene expression profiling in the reprogramming process

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mRNA profiling of genetically matched ESCs and iPSCs

Project description:Induced pluripotent stem cells (iPSCs) can be generated by enforced expression of defined transcription factors in somatic cells. It remains controversial whether iPSCs are equivalent to blastocyst-derived embryonic stem cells (ESCs). Using genetically matched cells, we found that the overall mRNA expression patterns of these cell types are indistinguishable with the exception of a few transcripts encoded on chromosome 12qF1. iPSCs were derived from somatic tissues of chimeric mice generated with ESCs that carry an inducible cassette encoding for the reprogramming factors myc, Klf4, Oct4 and Sox2.

2010-04-28 | E-GEOD-20572 | biostudies-arrayexpress

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Project description:We report the miRNA profiling in MEF cells, ES cells and three Pluripotent Stem Cells obtained by three different reprogramming approaches from MEF cells based on Solexa sequencing. iPS cells are reprogrammed by four factors (OSKM) from MEF cells. NT-ESCs were established by reprogramming MEF cells into ESCs using nuclear transfer. NT-iPSCs were established to reflect the combination of nuclear transfer and iPS technologies. iPSCs, NT-ESCs, and NT-iPSCs were exactly derived from the same MEF cells. The results indicate NT-ESCs give expression to the unique miRNAs other than both ESCs and iPSCs while pluripotent cells acquire or retain the pluripotent specific miRNAs compared with MEF. Furthermore, the comparison of different reprogramming cells suggests that several miRNAs have key roles in distinctly developmental potential reprogrammine cells. Small RNA profiles of MEF, ES, iPS, NT-ES and NT-iPS cells were generated by Solexa sequencing. MEF and ES cells were performed in triplicate. iPS, NT-ES and NT-iPS cells were sequenced in duplicate.

2014-07-05 | E-GEOD-52950 | biostudies-arrayexpress

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2013-11-11 | E-GEOD-46860 | biostudies-arrayexpress

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Project description:Expression of key transcription factors Klf4, Oct3/4, Sox2, and c-Myc (KOSM) in embryonic stem cells can reprogram somatic cells into pluripotent cells. We found that two histone variants, TH2A and TH2B, and histone chaperone Npm enhance the KOSM-dependent generation of induced pluripotent cells (iPSCs) and produce iPSCs only with Klf4 and Oct3/4. To identify directly affected genes by these histone variants during reprogramming, we carried out gene expression profiling of MEFs overexpressing TH2A/TH2B/Npm and TH2A/TH2B deficient MEFs after infection with retroviruses expressing KOSM. A total of 21 Affymetrix Mouse Gene ST array were done for mRNA expression profiling of ES cells, iPS cells induced by Klf4, Oct4, Sox2, and c-Myc (KOSM) or Klf4, Oct4, Th2a, Th2b, and p-Npm (KOBAN), wild-type MEFs infected with retrovirus vectors expressing KOSM, KOSMBAN, or empty vector and Th2a/Th2b-deficient MEFs infected with retrovirus vector expressing KOSM.

2013-12-25 | E-GEOD-53637 | biostudies-arrayexpress

Nanog Independent Reprogramming to iPSCs with Canonical Factors

Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line. 2-4 biological replicates each of 7 conditions (WT MEFs, WT ESC, WT iPSC, WT partially reprogrammed iPSC (piPS), Nanog null ESC, Nanog null iPSC clone G2 and Nanog null iPSC clone G5)

2014-01-01 | E-GEOD-53212 | biostudies-arrayexpress

Generation of Induced Pluripotent Stem Cells using Recombinant Proteins

Project description:Groundbreaking work demonstrated that ectopic expression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc, could reprogram murine somatic cells to induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006), and human iPSCs were subsequently generated using similar genetic manipulation (Takahashi et al., 2007,Yu et al., 2007). To address the safety issues arose from harboring integrated exogenous sequences in the target cell genome, a number of modified genetic methods have been developed and produced iPSCs with potentially reduced risks (for discussion, see Yamanaka, 2009, and references therein). However, all of the methods developed to date still involve the use of genetic materials and thus the potential for unexpected genetic modifications by the exogenous sequences in the target cells. Here we report generation of protein-induced pluripotent stem cells (piPSCs) from murine embryonic fibroblasts using recombinant cell-penetrating reprogramming proteins. We demonstrated that such piPSCs can long-term self-renew and are pluripotent in vitro and in vivo. Global gene-expression analyses of the piPS cells

2010-04-09 | E-GEOD-16062 | biostudies-arrayexpress

mRNA expression data from iPSCs, ntESCs and iPSC-nt-ESCs

Project description:We generated three kinds of genetically identical mouse reprogrammed cells: induced pluripotent stem cells (iPSCs), nuclear transfer embryonic stem cells (ntESCs) and iPSC-nt-ESCs that are established after successively reprogramming of iPSCs by nuclear transfer (NT). NtESCs show better developmental potential than iPSCs, whereas iPSC-nt-ESCs display worse developmental potential than iPSCs. We used microarrays to distinguish the gene expression differences among three pluriptoent stem cells and identified that imprinted genes had a similar expression pattern in iPSCs and iPSC-nt-ESCs. We sought to obtain genetic identical pluripotent stem cell lines in order to minimize genetic variations among different reprogrammed cells. To that end, we established a genetically homogenous secondary reprogramming system, in which mouse embryonic fibroblasts (MEFs) carrying doxycycline (dox)-inducible lentiviruses expressing Oct4, Sox2, Klf4 and c-Myc were isolated and used as donors for different reprogramming experiments. We generated iPSCs after plating MEFs in the presence of dox in ES culture conditions, and then derived ntESCs after transplantation of the nucleus from the same MEFs into enucleated oocyte. Furthermore, we successively reprogrammed iPSCs by means of NT and established a set of nt-iPSC lines. Pluripotent stem cells generated from different reprogramming strategies were for RNA extraction and hybridization on Affymetrix microarrays.

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