Stem Cell Reviews and Reports

, Volume 7, Issue 2, pp 331–341

A Cyclic AMP Analog, 8-Br-cAMP, Enhances the Induction of Pluripotency in Human Fibroblast Cells

Article

DOI: 10.1007/s12015-010-9209-3

Cite this article as:
Wang, Y. & Adjaye, J. Stem Cell Rev and Rep (2011) 7: 331. doi:10.1007/s12015-010-9209-3

Abstract

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However, the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3′, 5′-cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokine-related and inflammatory pathways. Remarkably, the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However, it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related, inflammatory pathways and self-renewal supporting gene, namely cyclin-encoding CCND2, and the associated genes CCNA1 and CCNE1. Conversely, we also see the down-regulation of the p53 (CCNB2, GTSE1, SERPINE1) and cell cycle (PLK1, CCNB2) pathways. Our data demonstrates that a cyclic AMP analog, 8-Br-cAMP, enhances the efficiency of cellular reprogramming. In addition, 8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming, which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming.

Keywords

Induced pluripotent stem cells Embryonic stem cells Pluripotency 8-Br-cAMP VPA p53 Cell cycle 

Supplementary material

12015_2010_9209_MOESM1_ESM.xls (121 kb)
Supplemental Table S1List of genes within each cluster generated from the comparison of the transcriptomes of HFF1, iPS, and ES (H1) cells. (XLS 121 kb)
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Supplemental Table S2List of genes commonly expressed in small molecule-treated cells and ES cells at day 1 of treatment. (XLS 105 kb)
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Supplemental Table S3List of genes commonly expressed in small molecule-treated cells and ES cells at day 7 of treatment. (XLS 90 kb)
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Supplemental Table S4List of genes involved in the differentially regulated pathways in small molecule-treated cells at day1 of treatment. (XLS 33 kb)
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Supplemental Table S5List of genes involved in the differentially regulated pathways in small molecule-treated cells at day 7 of treatment. (XLS 21 kb)
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Supplemental Table S6Primers for Quantitative Real-Time PCR and PCR reactions. (DOC 37 kb)
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Supplemental Fig. S1Optimization of transduction efficiency of retroviruses in HFF1 cells. GFP-encoding viruses were transduced into HFF1 cells using different viral concentrations in order to determine the optimal transduction efficiency of retrovirus. We chose as optimum the concentration of retrovirus with the highest transduction efficiency without obvious cell death. Approximately 60% of cells expressed GFP in the optimized concentration of retrovirus. The upper panel shows images taken with a fluorescent microscope. Scale bars represent 100 μm. The lower panel shows the results of flow cytometric analysis. (GIF 55 kb)
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Supplemental Fig. S2High resolution image. (TIFF 6842 kb)
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Supplemental Fig. S3Proliferation of HFF1 cells cultured in human ES cell culture medium with the indicated treatments. HFF1 cells were seeded on 24-well plates (2 × 104 cells/well). After 1 day, the cells were treated with 0.1 mM 8-Br-cAMP, 0.5 mM VPA, or 0.1 mM 8-Br-cAMP plus 0.5 mM VPA for 6 days. The medium was replaced every day. Proliferation was measured with alamar blue (AbD Serotec) at the indicated time points. Samples were analyzed in triplicate and the mean±standard deviation is shown. Comparison was performed between the different small molecule treatments and control at different time points. P values <0.05 are indicated by an asterisk. (GIF 142 kb)
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Supplemental Fig. S4High resolution image. (TIFF 818 kb)
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Supplemental Fig. S5iPS cells derived from 8-Br-cAMP-treated cells maintain a normal karyotype. GTG-banding karyotyping demonstrated iPS cells maintain a normal diploid male chromosomal content. (GIF 1 kb)
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Supplemental Fig. S6High resolution image. (TIFF 318 kb)
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Supplemental Fig. S7iPS cells were derived from their parent cell line. DNA fingerprinting analysis indicates that iPS cells were derived from their parental cell line (HFF1 cells) and not from contaminating ES cells (H1 and H9). (GIF 2 kb)
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Supplemental Fig. S8High resolution image. (TIFF 341 kb)
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Supplemental Fig. S9Differentiation potential of iPS cells in vivo. The representative series of hematoxylin-eosin stained sections from a teratoma produced from iPS-A4 show iPS cells differentiated into tissues representing all three embryonic germ layers, such as pigmented epithelium and neural tissue (ectoderm), cartilage (mesoderm), and tubular epithelium (endoderm). iPS-B4 also differentiated into derivatives of all three embryonic germ layers, including neural tissue (ectoderm), bone and cartilage (mesoderm), and epithelium with microvilli (endoderm). In contrast, iPS-C1 formed a cystic teratoma with gut-like epithelium (endoderm), spindle shaped stroma (mesoderm), and tubular epithelium (endoderm). All images of sections derived from each iPS cell line were obtained from the same tumor. Scale bars represent 100 μm. (GIF 222 kb)
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Supplemental Fig. S10High resolution image. (TIFF 6042 kb)
12015_2010_9209_MOESM17_ESM.jpg (63 kb)
Supplemental Fig. S11Venn diagram showing the distinct and overlapping transcriptional signatures between the different cell types. Left panel, day 1 of treatment. Right panel, day 7 of treatment. (JPEG 62.5 kb)
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Supplemental Fig. S12High resolution image. (TIFF 2.24 mb)

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Vertebrate Genomics, Molecular Embryology and Aging GroupMax Planck Institute for Molecular GeneticsBerlinGermany
  2. 2.The Stem Cell Unit, Department of Anatomy, College of MedicineKing Saud UniversityRiyadhSaudi Arabia