The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies ...have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney
. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages
. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.
Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of ...autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.
Blind enucleation is used in porcine somatic cell nuclear transfer (SCNT) to remove the metaphase II (MII) spindle from the oocyte. Deviation of the MII spindle location, however, leads to incomplete ...enucleation (IE). Here, we report that the rate of complete enucleation (CE) using the blind method was 80.2 ± 1.7%, although this significantly increased when the polar body-MII deviation was minimized (≦45°). While it is established that IE embryos will not survive to full term, the effect of IE on early stage development is unknown. We have previously demonstrated in mice and pigs that ETV2 deletion results in embryonic lethality due to the lack of hematoendothelial lineages. We observed that ETV2-null cloned embryos derived from blindly and incompletely enucleated oocytes had both WT and mutant sequences at E18 and, using FISH analysis, we observed triploidy. We also compared SCNT embryos generated from either CE or intentionally IE oocytes using the spindle viewer system. We observed a higher in vitro blastocyst rate in the IE versus the CE-SCNT embryos (31.9 ± 3.2% vs 21.0 ± 2.1%). Based on known processes in normal fertilization, we infer that the IE-SCNT embryos extruded the haploid second PB after fusion with donor fibroblasts and formed a near-triploid aneuploid nucleus in each blastomere. These studies demonstrate the peri-implantation survival of residual haploid nuclei following IE and emphasize the need for complete enucleation especially for the analysis of SCNT embryos in the peri-implantation stage and will, further, impact the field of reverse xenotransplantation.
PDF
