Generation of interspecies chimeras between primate induced pluripotent stem cells and porcine parthenogenetic embryos
The availability of human induced pluripotent stem cell (hiPSC) paves the way to generate regenerative tissue or organs from patient’s own stem cells. The production of chimeric piglets carrying organs that are entirely derived from human stem cells, or at least have a high contribution of human cells or tissues, could be used as a new tissue or an organ replacement in the future treatment of the patients. Here, we produced porcine-nonhuman primate chimeric embryos to assess the feasibility of the potential use of human iPSC for production of human stem cell-derived organs in pigs. Because in vitro culture conditions for cynomolgus monkey iPSC and porcine blastocysts are different, we first identified an effective in vitro culture system for porcine blastocysts and monkey iPSC. We compared blastocyst rates (Days 7 and 8) and number of cells of porcine parthenogenetic blastocysts (Day 8) cultured in 8 different monkey iPSC media and in porcine zygote medium 3 (PZM-3). The best developmental rates of porcine blastocysts were achieved in Knockout DMEM+20% serum replacement monkey medium (iPS 20% medium; N = 65, n = 3). The number of blastocysts on Day 8 cultured in iPS 20% medium was significantly higher (91%; P < 0.05) than in the commonly used porcine PZM-3 medium (65%). We found significantly fewer (P < 0.05) degenerate porcine embryos on Day 8 after culture in iPS 20% medium (9%) compared to PZM-3 (35%). The number of nuclei per blastocyst in iPS 20% medium (88 nuclei; N = 30, n = 3) was significantly higher (P < 0.0001) than in the PZM-3 medium (57 nuclei; N = 54, n = 3). Therefore, we decided to use iPS 20% medium for culture of porcine blastocysts injected with monkey iPSC. Thereafter, we injected clusters of 10 to 15 monkey iPSC transgenic with AAVS1-CAG-Venus into porcine parthenogenetic embryos from Days 4 and 6. Interspecies chimeras were cultured in iPS 20% medium for 24 (for Day 6 embryos) or 48 h (for Day 4 embryos) and observed by confocal microscopy to determine the proportion of Venus-expressing monkey iPSC in porcine embryos. Approximately 37% of blastocysts contained Venus-positive cells after injection of Day 6 embryos (N = 133, n = 4). In contrast, injection into porcine embryos from Day 4 resulted in 73% of Venus-positive blastocysts (N = 69, n = 3). Finally, we investigated proliferation and survival of monkey iPSC in interspecies chimeras after blastocyst plating onto murine fibroblasts. Chimeric blastocyst outgrowth resulted in Venus-expressing monkey iPSC proliferating over 1 week in culture. Outgrowths of all chimeric blastocysts established distinct but separate monkey and porcine stem cell colonies. Here, we optimized the culture conditions for an in vitro interspecies chimera assay in which monkey iPSC are able to survive in porcine embryos. Integration of monkey iPSC to host inner cell mass is relevant for the further contribution to the embryo development. Therefore, to verify this, analysis of cell-cell connection between monkey iPSC and porcine blastocysts and experiments using vivo-derived embryos are currently underway.
Nowak-Imiałek, Monika Anna / Wunderlich, S. / Herrmann, Doris / et al: Generation of interspecies chimeras between primate induced pluripotent stem cells and porcine parthenogenetic embryos. 2015.
Nutzung und Vervielfältigung:
Alle Rechte vorbehalten