297 PRODUCTION OF CHIMERIC PORCINE FETUSES BY AGGREGATION METHOD USING PARTHENOGENETIC EMBRYOS

Abstract

Porcine induced pluripotent stem (iPS) cells are considered to be an invaluable research tool in translational research with pigs as a large animal model. Pluripotency of the iPS cells needs to be verified by their competence to contribute to chimera formation. The aim of the present study is to establish feasible system to create chimeric pig fetuses using parthenogenetic embryos. In Experiment 1, inner cell mass (ICM) was isolated by immunosurgery from Day 6 blastocysts obtained by parthenogenetic activation of in vitro matured (IVM) oocytes. Isolated ICM were used as the donor cells after staining with fluorescent carbocyanine dye (DiI). Using parthenogenetic morulae or 4- to 8-cell embryos as the host embryos, chimeric embryos were prepared by injection or aggregation method. Injection of ICM was performed by micromanipulation: a single ICM was directly injected into the centre portion of the host morulae. In the aggregation method, a single ICM was aggregated with blastomeres isolated from 2 host embryos at the morula or 4- to 8-cell stage in a micro-well (400 µm diameter, 300 µm deep). The chimeric embryos were cultured in PZM-5 (Yoshioka et al. 2008) for 2 to 3 days to examine development to blastocysts and incorporation of donor ICM cells into the resultant blastocysts ICM (ICM chimerism). In Experiment 2, donor blastomeres isolated from a parthenogenetic morula or 4- to 8-cell embryo were stained by DiI and aggregated with a parthenogenetic host embryo at the morula or 4- to 8-cell stage, and the in vitro development to the blastocyst stage and the ICM chimerism were examined. In Experiment 3, ICM isolated from IVF blastocysts harboring humanized Kusabira-Orange (huKO) gene were used as donor cells. Donor ICM were aggregated with the host embryos at the morula or 4- to 8-cell stage, and the resultant blastocysts were transferred to 4 recipient gilts to collect fetuses on Day 18. Results of Experiments 1 and 2 are summarised in Table 1. Combination of the donor ICM and host morulae yielded high rates of blastocyst formation (~95%) and ICM chimerism (~85%), regardless of the method used (injection or aggregation). Transfer of 73 blastocysts developed from host morulae to 2 recipients (Experiment 3) gave rise to 25 (34.2%) fetuses, of which 6 (24.0%) were confirmed to be chimeric by their clear orange fluorescence and immunostaining by anti-huKO antibody. Of 22 (40.7%) fetuses obtained after transfer of 54 blastocysts derived from 4- to 8-cell host embryos to 2 recipients, 3 (13.6%) were chimeric. Contribution of the donor cells in the tissues of the chimeric fetuses measured by image analysis software (ImageJ, NIH, Bethesda, MD, USA) ranged between 16.1 and 65.2%. These results demonstrate that the aggregation method using parthenogenetic host embryos is an efficient means to produce chimeric pig fetuses, and thereby feasible for verification of pluripotent cells such as iPS cells.

Table 1.  In vitro development of injected or aggregated porcine embryos