Abstract
Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a “thymidine block”, but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.
Highlights
Human somatic cells can be reprogrammed by transducing defined factors into pluripotent stem cells, termed induced pluripotent stem cells [1,2]
We chose the elongation factor 1 α subunit (EF-1α) promoter that confers high levels of transgene expression in induced pluripotent stem cells (iPSCs) and neural stem/progenitor cells (NS/PCs), because we plan to use the herpes simplex virus type thymidine kinase (HSV-TK)/GCV system as a safety switch in iPSC-derived NS/PC transplantation for the treatment of spinal cord injury and as a suicide gene therapy for malignant glioma using iPSC-derived NS/PCs. iPSCs were transduced at a multiplicity of infection (MOI) of 5)
Transduced iPSCs were cultured under puromycin selection, and puromycin-resistant iPSCs were obtained at very low efficiency
Summary
Human somatic cells can be reprogrammed by transducing defined factors into pluripotent stem cells, termed induced pluripotent stem cells (iPSCs) [1,2]. Human iPSC technology has been widely used for disease modeling, drug discovery, and cell therapy development [3,4,5]. For applications in regenerative medicine, human iPSCs circumvent the ethical issues associated with human embryonic stem cells (ESCs). Autologous iPSCs or HLA-matched donor-derived iPSCs may overcome the problem of immune rejection. Genetic and epigenetic abnormalities occur during reprogramming and prolonged growth in cell culture [6,7]. A critical concern is the risk of tumorigenesis, when using iPSC-derived dividing cells such as neural stem/progenitor cells (NS/PCs) [8,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
