Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of Ccr4-Not complex as a regulator of transition from partial to genuine iPS cells

ABSTRACT: Partial induced pluripotent cells (iPSCs) are cell lines strayed from normal route from somatic cells to iPSCs and are immortalized. Mouse partial iPSCs are able to convert to real iPSCs by the exposure to 2i condition using MAPK and GSK3? inhibitors. However, the molecular mechanisms of this conversion are totally not known. Our piggyback vector mediated genome-wide screen revealed that Cnot2, one of core components of Ccr4-Not complex participates in this conversion. Subsequent analyses revealed other core components, i.e., Cnot1 and Cnot3 and Trim28 which is known to extensively share genomic binding sites with Cnot3 contribute to this conversion as well. Our bioinformatics analyses indicate that the major role of these factors in the conversion is the down-regulation of developmental genes in partial iPSCs. Two partial iPSC clones (2B1 and 5C5) cultured under conventional culture condition with leukemia inhibitory factor (LIF) and serum and those converted to iPSCs by the exposure to 2i condition were used for RNA source. Partial iPSCs (2B1) cultured under conventional condition in which either one of Cnot1, Cnot2, Cnot3 and Trim28 cDNA or these factors were combinatorially incorporated after retrovirus infection and those in which empty vector or Nanog were stably integrated were also used for RNA source. In addition, embryonic fibroblasts and embryonic stem cells (ESCs) from 13.5 dpc embryos and blastocysts, respectively, from Nanog-GFP transgenic mice and wild-type ESC line (EBRTcH3) were used for RNA source.

ORGANISM(S): Mus musculus

SUBMITTER: Akihiko Okuda

PROVIDER: E-GEOD-44339 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identification of Ccr4-not complex components as regulators of transition from partial to genuine induced pluripotent stem cells.

Kamon Masayoshi M Katano Miyuki M Hiraki-Kamon Keiko K Hishida Tomoaki T Nakachi Yutaka Y Mizuno Yosuke Y Okazaki Yasushi Y Suzuki Ayumu A Hirasaki Masataka M Ueda Atsushi A Nishimoto Masazumi M Kato Hidemasa H Okuda Akihiko A

Stem cells and development 20131209 18

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by defined factors. However, substantial cell numbers subjected to iPSC induction stray from the main reprogramming route and are immortalized as partial iPSCs. These partial iPSCs can become genuine iPSCs by exposure to the ground state condition. However, such conversion is only possible for mouse partial iPSCs, and it is not applicable to human cells. Moreover, the molecular basis of this conversion is completely unkn ...[more]

PMID: 24200330

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Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.

Dataset Information

Identification of Ccr4-Not complex as a regulator of transition from partial to genuine iPS cells

Publications

Identification of Ccr4-not complex components as regulators of transition from partial to genuine induced pluripotent stem cells.

Similar Datasets

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