Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human induced pluripotent stem (iPS) cells isolated from from neonatal skin derived cells

ABSTRACT: Induction of germline-competent pluripotent stem cells from mouse fibroblasts has been achieved by the ectopic expression of four genes (Oct3/4, Sox2, c-Myc and Klf4). If this method can be applied to humans for the generation of personalized human pluripotent stem cells, it would greatly facilitate the therapeutic application of stem cells by avoiding the problem of immune rejection by the recipient associated with allograft transplants. Here we show that the ectopic expression of the same four genes in human neonatal skin derived cells is sufficient to induce pluripotent stem cells indistinguishable from human embryonic stem cells in morphology, gene expression, DNA methylation, teratoma formation and long term self-renewal ability. Extensive analysis of colonies generated by ectopic expression of these four genes indicates the presence of considerable heterogeneity in the induced colonies. These results provide a new finding to generate human induced pluripotent stem cells from postnatal somatic tissues. Experiment Overall Design: The microarray study was carried out using Affymetrix Human Genome U133 Plus 2.0 gene expression arrays or Agilent Whole Human Genome Oligo microarrays. Total RNA was extracted from cells with RNeasy (Qiagen). For Affymetrix array, biotin-labelled cRNA was reverse transcribed from 1 Âµg of total RNA according to Affymetrix technical protocols. Fifteen micrograms of cRNA was fragmented and hybridized to Affymetrix U133 plus 2 GeneChip arrays at 45C for 16 h and then washed and stained using Affimetrix Fluidics. The arrays were scanned using an Affimetrix GCS3000 scanner, and the images obtained were processed to data using the GCOS software. For Agilent array (G4112A), Total RNA was labeled with Cy3. Samples were hybridized with Agilent Gene Expression Hybridization kit according to the one color protocol. Arrays were scanned with a Agilent Microarray Scanner System and processed to data with Feature Extraction Software (v.9.5.3). Data from this experiment and the GEO database were analysed with the GeneSpring 7.3.1. software.

ORGANISM(S): Homo sapiens

SUBMITTER: Tetsuya Ishikawa

PROVIDER: E-GEOD-9709 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility to generate patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Considering that ectopic expression of c-Myc causes tumourigenicity in offspring and retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here, we show that adult mouse neural stem cells (NSCs) express higher endogenous levels of Sox2 and c-Myc than ES cells and that exogenous Oct4 together with either Klf4 or c-Myc are sufficient to generate iPS cells from NSCs. These two-factor (2F) iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germline, and form chimeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors. Experiment Overall Design: 8 hybridizations in total. Experiment Overall Design: NSC derived iPS cells by 2 factors (Oct4 and Klf4) in triplicate: Experiment Overall Design: - iPS cell_2F_1 Experiment Overall Design: - iPS cell_2F_2 Experiment Overall Design: - iPS cell_2F_3 Experiment Overall Design: Embryonic Stem cells (ESC) in triplicate: Experiment Overall Design: - ESC_1 Experiment Overall Design: - ESC_2 Experiment Overall Design: - ESC_3 Experiment Overall Design: NSC cultures in duplicates: Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: NSC derived iPS cells by 4 factors (Oct4, Sox2, c-Myc and Klf4) in triplicate: Experiment Overall Design: - iPS cell_4F_1 Experiment Overall Design: - iPS cell_4F_2 Experiment Overall Design: - iPS cell_4F_3

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Transcription profiling of human induced pluripotent stem (iPS) cells isolated from from neonatal skin derived cells

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