జర్నల్ ఆఫ్ స్టెమ్ సెల్ రీసెర్చ్ & థెరపీ

నైరూప్య

Successful Induction of Pluripotent Stem Cells From a Fabry Disease Mouse Model: Toward the Development of Safe Lentiviral Gene Therapy

Makoto Yoshimitsu, Koji Higuchi, Naomichi Arima, Jeffrey A Medin and Toshihiro Takenaka

Induced pluripotent stem (iPS) cells are now recognized as a valuable tool for cell repair through autologous transplantation. These cells are obtainable from somatic cells through the induction of the transcriptional factors Oct- 3/4, Klf4, and Sox2. In this study, we successfully established iPS cells from the tail-tip fibroblasts of a α-galactosidase A-knockout mouse, a well-known Fabry disease mouse model. These Fabry-iPS cells exhibited an embryonic stem (ES) cell phenotype, characterized by the expression of SSEA-1, increased alkaline phosphatase activity, silencing of the retroviral-transgene, and embryoid body (EB) formation. Subcutaneous inoculation of Fabry-iPS cells into nude mice resulted in teratoma formation. At day 6, EBs in differentiation media showed hematopoietic lineage-specific gene expression. In addition, we observed spontaneous contraction of EBs cultured on OP9 stroma cells for 5-7 days. RT-PCR demonstrated that various cardiac marker genes, such as Nkx2.5, Gata4, Tnnt2 (cardiac troponin T), and Mlc2a were more highly expressed in differentiation cultures of Fabry iPS cells than of control feeder cells. To assess their potential use for gene therapy, lentiviral transduction of Fabry-iPS cells with α-galactosidase-A cDNA, the therapeutic gene for Fabry disease, was performed. This transduction resulted in elevated intracellular and secreted α-galactosidase A activity. The ES cell-specific gene expression profile remained unaltered by lentiviral therapeutic gene transfer for more than 30 days post-transduction. These findings demonstrate that Fabry-iPS cells are readily obtainable and amenable for use in gene therapy.