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Noninvasive Reporter Gene Imaging of Human Oct4 (Pluripotency) Dynamics During the Differentiation of Embryonic Stem Cells in Living Subjects

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Abstract

Purpose

Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects.

Procedures

To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter.

Results

In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) (p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h (p = 0.001) and between 12 and 168 h (p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments (n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers.

Conclusions

We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects.

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Abbreviations

ES cells:

Embryonic stem cells

hfluc2:

Humanized firefly luciferase gene

hrluc:

Humanized Renilla luciferase gene

pOct4:

Oct4 promoter

pUbi:

Ubiquitin promoter

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Acknowledgments

This work is supported by National Cancer Institute In living subjects Cellular and Molecular Imaging Center grant P50 CA 114747 (S.S.G), GE global research program (S.S.G). (B.A), was supported by the Nuclear Infra Construction of Nuclear R & D program, Korean ministry of Science and Technology. (N.P.) was supported by the Doris Duke Foundation, Stanford Dean’s Fellowship, and Stanford NIBIB T32 Training grant EB009035.

Author Disclosure Statement

All authors declare that they have no potential conflict of interest in connection with the submitted article.

Author information

Author notes

  1. Natesh Parashurama

    Present address: Institute of Regenerative Medicine and Stem Cells, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco (UCSF) School of Medicine, RMB 902 E, 35 Medical Center Way, San Francisco, CA, 94143, USA

Authors and Affiliations

  1. Molecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford University, 318 Campus Drive, E153, Stanford, CA, 94305-5427, USA

    Byeong-Cheol Ahn, Natesh Parashurama, Manish Patel, Keren Ziv, Shahriar Shah Yaghoubi, Ramasamy Paulmurugan & Sanjiv Sam Gambhir

  2. Department of Nuclear Medicine, Kyungpook National University Medical School, Kyungpook National University Hospital, 50 Samduck 2Ga, Chung Gu, Daegu, 700-712, South Korea

    Byeong-Cheol Ahn

  3. GE Global Research Center, General Electric, Niskayuna, NY, USA

    Srabani Bhaumik

  4. Canary Center for Early Detection of Cancer, Palo Alto, CA, USA

    Ramasamy Paulmurugan & Sanjiv Sam Gambhir

  5. Department of Bioengineering, Stanford School of Medicine, Stanford, CA, USA

    Sanjiv Sam Gambhir

  6. Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA

    Sanjiv Sam Gambhir

Authors
  1. Byeong-Cheol Ahn
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Corresponding author

Correspondence to Sanjiv Sam Gambhir.

Additional information

Byeong-Cheol Ahn and Natesh Parashurama contributed equally to this work.

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Ahn, BC., Parashurama, N., Patel, M. et al. Noninvasive Reporter Gene Imaging of Human Oct4 (Pluripotency) Dynamics During the Differentiation of Embryonic Stem Cells in Living Subjects. Mol Imaging Biol 16, 865–876 (2014). https://doi.org/10.1007/s11307-014-0744-1

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  • DOI: https://doi.org/10.1007/s11307-014-0744-1

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