Dataset Information

Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

ABSTRACT:

Background

The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two types of iPSC and of partially reprogrammed iPSCs (iPSCp) generated independently from adult cardiac and skin fibroblasts to assess any perturbations of the transcription program during reprogramming.

Results

The comparative dissection of the transcription profiles and histone modification patterns at lysines 4 and 27 of histone H3 of the iPSC, iPSCp, ESC, and somatic cells revealed that the iPSC was almost completely comparable to the ESC, regardless of their origins, whereas the genes of the iPSCp were dysregulated to a larger extent. Regardless of the origins of the somatic cells, the fibroblasts induced using the ESC protein extracts appear to be completely reprogrammed into pluripotent cells, although they show unshared marginal differences in their gene expression programs, which may not affect the maintenance of stemness. A comparative investigation of the iPSCp generated by unwanted reprogramming showed that the two groups of genes on the pathway from somatic cells to iPSC might function as sequential reprogramming-competent early and late responders to the induction stimulus. Moreover, some of the divergent genes expressed only in the iPSCp were associated with many tumor-related pathways.

Conclusions

Faithful transcriptional reprogramming should follow epigenetic alterations to generate induced pluripotent stem cells from somatic cells. This genome-wide comparison enabled us to define the early and late responder genes during the cell reprogramming process to iPSC. Our results indicate that the cellular responsiveness to external stimuli should be pre-determined and sequentially orchestrated through the tight modulation of the chromatin environment during cell reprogramming to prevent unexpected reprogramming.

SUBMITTER: Park J

PROVIDER: S-EPMC5289558 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

Park Jihwan J Kwon Yoo-Wook YW Ham Seokjin S Hong Chang-Pyo CP Seo Seonghye S Choe Moon Kyung MK Shin So-I SI Lee Choon-Soo CS Kim Hyo-Soo HS Roh Tae-Young TY

PloS one 20170202 2

<h4>Background</h4>The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two type ...[more]

PMID: 28152015

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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.

Dataset Information

Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

Background

Results

Conclusions

Publications

Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

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