Research Article

Molecular Imaging and Biology

, Volume 16, Issue 6, pp 865-876

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

  • Byeong-Cheol AhnAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford UniversityDepartment of Nuclear Medicine, Kyungpook National University Medical School, Kyungpook National University Hospital
  • , Natesh ParashuramaAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford UniversityInstitute of Regenerative Medicine and Stem Cells, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco (UCSF) School of Medicine
  • , Manish PatelAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford University
  • , Keren ZivAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford University
  • , Srabani BhaumikAffiliated withGE Global Research Center, General Electric
  • , Shahriar Shah YaghoubiAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford University
  • , Ramasamy PaulmuruganAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford UniversityCanary Center for Early Detection of Cancer
  • , Sanjiv Sam GambhirAffiliated withMolecular Imaging Program @Stanford (MIPS), Department of Radiology, Division of Nuclear Medicine, James H. Clark Center, Stanford School of Medicine, Stanford UniversityCanary Center for Early Detection of CancerDepartment of Bioengineering, Stanford School of MedicineDepartment of Materials Science and Engineering, Stanford University Email author 

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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.

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