Since human embryonic stem (ES) cell lines have the ability to both proliferate indefinitely and differentiate into multiple tissue cells, they are expected to have clinical applications as well as to serve as models for basic research and drug development. Although efficient and stable gene transfer into primate ES cells would be useful for such purposes, it has been difficult and only lentiviral vectors have been successful in achieving it. We have previously developed Sendai virus (SeV) vector that is capable of self-replication but incapable of transmitting to other cells. The vector replicates in the form of negative-sense single-stranded RNA in the cytoplasm of infected cells and does not go through a DNA phase. SeV vector can efficiently introduce foreign genes without toxicity into various types of cells including hematopoietic progenitor cells. In this study, we have examined the efficacy of SeV vector to introduce a new genetic material into nonhuman primate cynomolgus ES cells.

Cynomolgus ES cells were once exposed to a SeV vector carrying the GFP gene (50 TU/ml). About 60% of cells stably fluoresced at least for a month as assessed by flow cytometry. Fluorescent ES-cell colonies were plucked under a fluorescent microscope once at one month after infection and they were propagated. The GFP gene was then vigorously and stably expressed in about 90% of the cells for a year. The undifferentiated cell fractions were unchanged after the SeV infection of ES cells as assessed by staining with alkaline phosphatase and SSEA-4. In addition, the infected, fluorescent ES cells were able to form fluorescent teratomas when transplanted into immunodeficient mice. Thus, the SeV infection did not hamper pluripotency of ES cells and transgene silencing did not occur during the teratoma formation in vivo. For induction of hematopoietic differentiation, the infected, fluorescent ES cells were seeded onto OP9 stromal cells in the presence of multiple cytokines including BMP-4 and VEGF. Cells at day 18 were subjected to clonogenic hematopoietic colony formation assays. Resulting colonies at day 14 fluoresced and the colonies contained NBT-positive, fluorescent mature granulocytes. Thus, SeV-mediated transgene was stably expressed even after the terminal differentiation to granulocytes. In addition, SeV-mediated GFP expression levels in ES cells could be reduced dose-dependently by the addition of an anti-RNA virus drug, ribavirin, to the culture. Upon discontinuation of ribavirin treatment, the cells regained the original level of GFP expression. Ribavirin is an inhibitor of viral RNA polymerase and used for treatment of hepatitis C infection. In conclusion, SeV vector will be a useful tool for gene transfer into primate ES cells and the method of using anti-viral drugs should allow further investigation for regulated SeV-mediated gene expression.

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