Noninvasive reporter gene imaging of human Oct4 (pluripotency) dynamics during the differentiation of embryonic stem cells in living subjects.
OBJECTIVE
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.


METHODS
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.
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, abc2000@knu.ac.kr.
Byeong-Cheol
BC
Ahn
Natesh
N
Parashurama
Manish
M
Patel
Keren
K
Ziv
Srabani
S
Bhaumik
Shahriar Shah
SS
Yaghoubi
Ramasamy
R
Paulmurugan
Sanjiv Sam
SS
Gambhir
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D039904:Pluripotent Stem Cells; D051381:Rats
D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D008156:Luciferases