ASCB Abstract: Increased rate of reprogramming of induced pluripotent stem cells using high-titer lentiviral vectors encoding multiple cell growth and survival regulatory genes

Liang Yin, Jiwu Wang, Lung-Ji Chang and Yichen Wang

Objective: Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSC) with enforced expression of multiple transcription factors. We aim to improve the reprogramming efficiency using high titer lentiviral vectors encoding additional cell growth and survival regulatory genes.

Methods: Lentiviral vectors encoding multiple cell cycle and apoptosis genes in addition to c-Myc, Klf4, Oct4 and Sox2 were constructed and used to generate iPSC. The iPSC were extensively characterized by immunohistochemical staining and flow cytometry.

Results: Human mesenchymal stem cells can be efficiently transduced and reprogrammed into iPSC using high-titer lentiviral vectors encoding the four known transcription factors. The addition of siRNA suppressing p53 and cell cycle and survival genes including telomerase and BclXL significantly increased the efficiency and rate of iPSC generation. Human iPSC colonies were formed within a week after lentiviral gene transfer.

Conclusions: The protocol for iPSC generation has been improved with high titer lentiviral vectors encoding additional immortalization cellular factors regulating cell cycle progression, senescence and apoptosis. Deletion of the integrated lentiviral genomes using Cre-loxP recombination could increase the safety profile of the reprogrammed iPSC.

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