118 INDUCING PLURIPOTENCY IN SOMATIC CELLS FROM THE SNOW LEOPARD (PANTHERA UNCIA), AN ENDANGERED FELID

Abstract

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMX) encoding either 4 (Oct-4, Sox-2, Klf-4 and cMyc) or 5 (Oct-4, Sox-2, Klf-4, cMyc and Nanog) human transcription factors. Transduction efficiency of the retrovirus was ascertained using pMX-green fluorescent protein transgene expression and averaged 96% from 3 repeated experiments. The reprogramming efficiency of initial colony formation was 0.000308% (37/120 000 cells plated) for 4-factor induction compared with 0.000517% (62/120 000) for 5-factor induction. Transduction with 4 factors resulted in the formation of small colonies of cells, which could not be maintained for more than 4 passages (P4). However, addition of Nanog to the transfection cocktail produced stable iPS cell colonies, which formed as early as Day 3. Colonies of cells were selected at Day 5 and expanded in vitro on mouse embryonic fibroblast feeder cells. The resulting cell line was positive for alkaline phosphatase, Oct-4, Nanog and stage-specific embryonic antigen-4 at P14. Also, RT-PCR confirmed that endogenous Oct-4 and Nanog were expressed by snow leopard iPS cells from P4; although all 5 human transgenes were transcribed at P4, Oct-4, Sox-2 and Nanog transgenes were silenced as early as P14, suggesting that reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the 3 germ layers. This study describes the first derivation of iPS cells from the endangered snow leopard and is also the first report on induced pluripotency in felid species. Our results demonstrate that addition of Nanog to the reprogramming cocktail was essential for derivation of iPS lines in this felid and that iPS cells provide a unique source of pluripotent cells with utility in conservation for cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.