The differentiation of individual induced pluripotent stem cells (hiPSC) to generate specific types of cells is inefficient and the remaining undifferentiated cells may form teratomas. prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN) and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of GREM1 the nine puromycin-resistant SGC-CBP30 iPSC clones analyzed three contained the HSV1-TK transgene at the OCT4 locus but they were not sensitive to GCV. The other six clones were GCV-sensitive but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed. Introduction Human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (hESC) have the capacity to proliferate indefinitely and to differentiate into SGC-CBP30 cells of all three germ layers. Thus they provide promising resources for regenerative medicine and other applications such as disease modeling and drug screening. HiPSCs are SGC-CBP30 attractive because autologous cellular products can be derived from a patient’s own iPSC minimizing the risk of immunogenicity and graft rejection SGC-CBP30 and because hiPSC can be derived from somatic cells SGC-CBP30 avoiding the need to destroy embryos. However recent reports have raised concerns regarding the safety of these cells for clinical applications . For example the differentiation of hiPSC to generate specific types of cells is inefficient and the remaining undifferentiated cells may form teratomas   . A number of strategies have been explored to decrease the presence of undifferentiated cells and to mitigate the risk of teratoma formation. These strategies include extending the time of differentiation   use of positive and/or negative selection markers      and suicide genes. The herpes simplex virus 1 thymidine kinase (HSV1-TK) gene is a commonly used suicide gene for ESC     . In addition to ESC the HSV1-TK suicide gene has also been tested SGC-CBP30 in the context of iPSC    . Ganciclovir (GCV) the prodrug of HSV1-TK can be converted to cytotoxic GCV-triphosphate by HSV1-TK thereby killing HSV1-TK-expressing cells. In some of the studies reported teratomas still formed after engraftment even though the stem cells had been engineered to express HSV1-TK and the cellular product had been treated with GCV  . One possible reason for this outcome is that the TK gene in the engineered cells mutated over time a frequent observation for HSV1-TK-expressing tumor cells    . In addition promoter silencing may have been an issue . In this study we attempted to site-specifically insert a HSV1-TK transgene sequence into the OCT4 locus of hiPSC using an OCT4-specific ZFN pair (OCT4 ZFN.
- Hematopoietic stem cell (HSC) therapy is usually widely used to treat a growing number of hematological and non-hematological diseases
- Supplementary Materials? JCMM-23-2943-s001
- Supplementary MaterialsFIGURE S1: Characterization of the IMFNCR Series
- Supplementary MaterialsSupplementary data
- Body fat grafting procedures are believed to be always a appealing regenerative, cell\directed therapy; nevertheless, their survival is influenced by ischemia condition
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