Background: Human dermal fibroblasts (HDFs) from older subjects are known to be more resistant to reprogramming. Results: Inclusion of SIRT6 can significantly improve the reprogramming efficiency. Conclusion: Changes in SIRT6 expression and its posttranscriptional regulation may be relevant in aging. Significance: MiR-766-mediated posttranscriptional regulation of SIRT6 has implications in human aging.
Rationale
Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied upon random integration, a method that is associated with unwanted insertional mutagenesis and position effects on transgene expression.
Objective
To address this barrier, we used genome editing with zinc finger nuclease technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) for molecular imaging.
Methods and Results
We employed ZFN technology to integrate a construct containing monomeric red fluorescent protein (mRFP), firefly luciferase (Fluc), and herpes simplex virus thymidine kinase (HSVtk) reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for bioluminescence imaging (BLI) and positron emission tomography (PET) imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both hESCs and iPSCs. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited hESCs, iPSCs, and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the utility of ZFN-edited cells for preclinical studies in regenerative medicine.
Conclusion
Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells (PSCs) and their progeny for molecular imaging in vitro and in vivo.
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