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Molecular Imaging and Biology

, Volume 16, Issue 6, pp 865–876 | Cite as

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

  • Byeong-Cheol Ahn
  • Natesh Parashurama
  • Manish Patel
  • Keren Ziv
  • Srabani Bhaumik
  • Shahriar Shah Yaghoubi
  • Ramasamy Paulmurugan
  • Sanjiv Sam GambhirEmail author
Research Article
First Online:

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.

Key words

Embryonic stem cells Pluripotent stem cells Differentiation Human Oct4 Reporter gene imaging Gene networks Molecular imaging Oct4 Pluripotency In vivo imaging In vivo differentiation 

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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Notes

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.

Supplementary material

11307_2014_744_MOESM1_ESM.pdf (7.1 mb)
ESM 1 (PDF 7220 kb)

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Copyright information

© World Molecular Imaging Society 2014

Authors and Affiliations

  • Byeong-Cheol Ahn
    • 1
    • 2
  • Natesh Parashurama
    • 1
    • 7
  • Manish Patel
    • 1
  • Keren Ziv
    • 1
  • Srabani Bhaumik
    • 3
  • Shahriar Shah Yaghoubi
    • 1
  • Ramasamy Paulmurugan
    • 1
    • 4
  • Sanjiv Sam Gambhir
    • 1
    • 4
    • 5
    • 6
    Email author
  1. 1.Molecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark CenterStanford School of Medicine, Stanford UniversityStanfordUSA
  2. 2.Department of Nuclear MedicineKyungpook National University Medical School, Kyungpook National University HospitalDaeguSouth Korea
  3. 3.GE Global Research Center, General ElectricNiskayunaUSA
  4. 4.Canary Center for Early Detection of CancerPalo AltoUSA
  5. 5.Department of BioengineeringStanford School of MedicineStanfordUSA
  6. 6.Department of Materials Science and EngineeringStanford UniversityStanfordUSA
  7. 7.Institute of Regenerative Medicine and Stem Cells, Department of Obstetrics, Gynecology, and Reproductive SciencesUniversity of California San Francisco (UCSF) School of MedicineSan FranciscoUSA

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