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The polycomb protein Ezh2 impacts on induced pluripotent stem cell generation.

ABSTRACT: Reprogramming of somatic cells toward pluripotency involves extensive chromatin reorganization and changes in gene expression. Polycomb group (PcG) proteins are key regulators of chromatin structure, cell identity, and development. In this study, we investigated the impact of Ezh2, a core subunit of Polycomb repressive complex 2 (PRC2), on the generation of induced pluripotent stem (iPS) cells. We found that Ezh2 expression is induced during iPS cell generation and iPS cells contain high levels of Ezh2 mRNA and protein. Importantly, shRNA knockdown of Ezh2 during reprogramming severely impairs iPS cell generation. Mechanistically, Ezh2 acts during reprogramming at least in part through repressing the Ink4a/Arf locus, which represents a major roadblock for iPS cell generation. Interestingly, knockdown of Ezh2 in established pluripotent cells leaves pluripotency and self-renewal of embryonic stem cells and iPS cells unaffected. Altogether, our results demonstrate that Ezh2 is critical for efficient iPS cell generation, whereas it is dispensable for maintaining the reprogrammed iPS cell state.

SUBMITTER: Ding X

PROVIDER: S-EPMC3996971 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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The polycomb protein Ezh2 impacts on induced pluripotent stem cell generation.

Ding Xiaolei X Wang Xiaoying X Sontag Stephanie S Qin Jie J Wanek Paul P Lin Qiong Q Zenke Martin M

Stem cells and development 20140121 9

Reprogramming of somatic cells toward pluripotency involves extensive chromatin reorganization and changes in gene expression. Polycomb group (PcG) proteins are key regulators of chromatin structure, cell identity, and development. In this study, we investigated the impact of Ezh2, a core subunit of Polycomb repressive complex 2 (PRC2), on the generation of induced pluripotent stem (iPS) cells. We found that Ezh2 expression is induced during iPS cell generation and iPS cells contain high levels ...[more]

PMID: 24325319

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Project description:Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-h intervals, and cultured on mitomycin C treated mouse embryonic fibroblast (MEF) cells in complete embryonic stem cell (ESC) medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to ESCs and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by reverse transcription-polymerase chain reaction. The protein iPSCs (piPSCs) formed embryoid bodies and subsequently differentiated towards all three germ layer lineages. Importantly, the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming MEFs to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESCs: form round colonies with defined boundaries; have a tendency to attach together with high nuclear/cytoplasmic ratio; express key pluripotency markers; and are capable of in vitro differentiation into ecto-, endo-, and mesoderm, and in vivo chimera formation.

Project description:BackgroundPigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.MethodsiPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.ResultsIn this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.ConclusionsOur findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

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The polycomb protein Ezh2 impacts on induced pluripotent stem cell generation.

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The polycomb protein Ezh2 impacts on induced pluripotent stem cell generation.

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