326 ASSESSMENT OF PORCINE-INDUCED PLURIPOTENT STEM CELLS BY IN VIVO ASSAYS

Abstract

Porcine-induced pluripotent stem cells (piPSC) have been established since 2009, but only 1 report demonstrated contribution to germline chimeras. One well-established in vivo pluripotency assay is the teratoma assay, which has recently been questioned due to the lack of standardized guidelines. In the present study we have characterised GFP-tagged in vitro and in vivo tetracycline-inducible piPSC [porcine MYC, SOX2, KLF4 (pOMSK)] and their capacity to form teratomas. We injected 1.5 million cells in 250 µL of PBS subcutaneous into NOD/SCID mice and followed them up to 6 weeks. The teratomas were analysed by immunohistochemistry for the 3 germlayer markers β3 tubulin, α fetoprotein, and smooth muscle actin. We not only found our teratomas positive for these markers, but also co-positive for GFP, clearly showing that the teratoma was made from porcine cells, which was not sufficiently proven in former studies. Our H&E staining revealed the following structures: cuboidal ephitelium, thyroid-like structure, renal corpuscle, and steroid producing cells. We continued to test the capacity of our venus iPS cells to contribute to in vitro chimeras. To achieve this we used a micromanipulator to inject 15 cells into Day 5 parthenotes, and subsequently cultured them in PZM3 with 10% FCS, cultured with or without doxycycline. These in vitro chimeras were followed until Day 7 in Nikons Biostation IM and used for differential staining. In all groups we saw good survival, hatching, and maintenance of GFP, indicating integration of these cells in our in vitro assay. We only found differences between survivals of the cell lines in the group cultured with doxycycline. Finally, in order to assess if the naïve type venus iPS cells could possibly be a truly naïve piPSC, we tested their capacity to form in vivo germline chimeras. This was tested by injecting 15 piPSC into Day 4 to 5 in vivo embryos. The injected embryos were transferred into 5 surrogate mothers, 3 of them were fed doxycycline before the transfer and 5 days after, and the last 2 recipient sows were not fed doxycycline. The pregnancies were terminated at Day 32 and the embryos were examined for fluorescence and the GFP transgene by PCR. In summary, it appears that both naïve type and primed type venus iPS cells are still strongly dependent on the pOMSK transgene expression, and the ultimate proof for pluripotency, the chimera production, seems to be not achievable under the condition we have chosen.