101 TOWARDS OPTIMAL IN VITRO CULTURE CONDITIONS FOR PIG-MONKEY AGGREGATION CHIMERAS

Abstract

Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical treatments. One potential use of iPSCs is for xenotransplantation via production of chimeric organisms. The generation of organs originated from the iPSCs of one individual in a foreign organism could allow the production of immune compatible organ transplants. One of the major problems down this road is to define the different nutrient needs of chimeric embryos (i.e. iPSCs and the host embryos). Here, we evaluated different media for supporting development of chimeric embryos consisting of parthenogenetic porcine embryos and iPS cells, either from pig or nonhuman primate, to identify the optimal medium conditions for pig-monkey aggregation chimeras. First, we cultured 3-day-old porcine parthenogenetic embryos in porcine zygote medium (PZM), iPSC-medium, and mixtures of the two media to identify the most suitable culture conditions. Three-day-old parthenogenetic embryos developed poorly in pure iPSC-medium (6.3% blastocyst rate), but grew in a mixture of PZM and iPSC medium. The best results were achieved with PZM and PZM with 10% or 25% iPSC medium (38.8% and 30% blastocyst rates, respectively). Next, we checked aggregation results of chimeric embryos produced with two different iPSC lines in the respective media (PZM, PZM+10% iPSC medium, PZM+25% iPSC medium). The porcine iPSC line (piPSC) and cynomolgus monkey iPSC line (ciPSC) carry fluorescent markers (piPSC: GFP, ciPSC: venus), thus facilitating detection of integration into the host embryos. After aggregation of iPS cells between two Day-3 parthenogenetic embryos (sandwich technique), blastocyst rates at Day 6 were determined (see Table 1). While culture in PZM allowed for the highest blastocyst rate (>40%), the iPS cell participation was very low (0–11.1%). Cultivation in PZM+25% iPS medium was compatible with a high rate of embryo-iPSC chimeras (50–100%), but with a low blastocyst rate (7.7–16.7%). Possibly, the iPS cells proliferate more rapidly than blastomeres and the embryo is overgrown. The best results were obtained in medium mixture PZM+10% iPSC medium (25–40% blastocyst rate with 66.7–100% iPS cell participation). These results show that supplementation of the basic culture medium with 10% iPSC medium yields high blastocyst rates for chimeric embryos, and also ensures much higher iPS cell participation. This improves the use of 3-day-old embryos in aggregation chimeras.

Table 1.  Development of parthenogenetic porcine embryo aggregates with porcine (p) or cynomolgus monkey (c) iPS cells