Discussion

Induced pluripotent stem cells (iPSCs) are valuable research tools for many different applications, including drug discovery, disease modeling, and regenerative medicine. Given the importance and potential of iPSC, it is vital that researchers have access to reprogramming methods that can safely and efficiently generate iPSCs from a wide variety of patient-derived cell types. The CytoTune®-iPS Sendai Reprogramming kit has emerged as an efficient method for generation of footprint-free iPSC from a wide variety of cell types, including blood cells. However, issues that were observed in some donor cells included toxicity, low reprogramming efficiency, and persistence of the virus beyond passage 10.
To address the above challenges, novel configurations of the virus were identified and used for the development of the CytoTune®-iPS 2.0 Sendai Reprogramming Kit. The resulting new configuration consists of the Oct4, Sox2, and Klf4 genes in a single polycistronic vector in a new temperature sensitive backbone, with c-Myc remaining on a separate vector, and an additional separate Klf4 vector used to enhance efficiency. The polycistronic vector was designed for greater efficiency of reprogramming in a temperature sensitive backbone that allows for lower cytotoxicity and faster viral clearance. The additional Klf4 vector further aids in achieving maximum efficiency with minimal viral load. Consistent with this, CytoTune® 2.0 was found to have 2-5 fold greater efficiency of reprogramming in fibroblast and blood reprogramming. In addition, the level of toxicity in primary cells was significantly reduced with CytoTune® 2.0, relative to the previous version. Clones derived with CytoTune® 2.0 had positive pluripotent marker expression, tri-lineage differentiation potential, and normal karyotype. In addition, the viral backbone was diluted from the majority of clones as early as passage 3. Thus, the CytoTune 2.0®-iPS Sendai Reprogramming kit provides a robust system for higher efficiency, low toxicity, and faster viral clearance from resulting iPSC clones.