SUMMARY Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Surgical procedures are prevalent in porcine embryo transfer (ET) programs, where the use of vitrified embryos is quasi non-existent. This study compared the effectiveness of surgical vs nonsurgical deep uterine (NsDU) ET using vitrified, in vivo-derived embryos (morulae and blastocysts) on the reproductive performance and welfare of the recipients. The recipient sows (n = 122) were randomly assigned to one of the following groups: surgical ET with 30 vitrified-warmed embryos (S-30 group, control); NsDU-ET with 30 vitrified-warmed embryos (NsDU-30 group) and NsDU-ET with 40 vitrified-warmed embryos (NsDU-40 group). Regardless of embryo stage, the NsDU-ET with 40 embryos presented similar rates of farrowing (72.7%) and litter size (9.9 ± 2.1 piglets) as the customary surgical procedure (75.0% and 9.6 ± 2.7 piglets). Numbers of ET-embryos appeared relevant, since the NsDU-ET with 30 embryos resulted in a decrease (P < 0.05) in farrowing rates (38.9%) and litter sizes (5.7 ± 2.4 piglets). In conclusion, we demonstrate for the first time that farrowing rate and litter size following a NsDU-ET procedure increase in function of a larger number of transferred vitrified embryos, with fertility equalizing that obtained with the invasive surgical approach. The results open new possibilities for the widespread use of non-invasive ET in pigs.

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The advancement of porcine embryo transfer (ET) technology is constrained by regulatory hurdles (liquid nitrogen transportation) or, more importantly, the technical obstacles of using vitrified embryos in combination with nonsurgical deep uterine ET technology. Maintaining embryos in culture during transport and prior ET collides with the need of CO gassing and the best choice of culture medium. In this work, we describe storage conditions for short-term embryo CO-free storage that allowed for a majority of in vivo-derived porcine morulae to survive after 3 days of storage in a liquid state, and to develop to the blastocyst stage unhatched, a sanitary prerequisite for ET. The storage conditions included NCSU-23 medium supplemented with bovine serum albumin, where bicarbonate was partially replaced by HEPES to avoid the need for CO gassing, and a temperature of 37 °C. These conditions were able to maintain the functionality of the stored embryos (hatching capacity after exposure to conventional culture conditions) and their developmental competence after ET (normal fetuses by day 38 of pregnancy). Use of this strategy of CO-free storage should allow the shipment of fresh embryos worldwide in the absence of liquid nitrogen.

The use of pH-stable media would simplify embryo vitrification and the warming of porcine embryos and might facilitate the application of embryo transfer in practice. In this work, we investigated whether a pH-stable basal medium constituted of Tyrode’s lactate medium, polyvinyl alcohol, and HEPES for buffering was suitable for porcine embryo vitrification warming in place of the conventional gas-equilibrated media. A high percentage (>90%) of embryos survived vitrification and warming in this medium, achieving in vitro survival rates similar to embryos vitrified-warmed using the conventional protocol and their fresh counterparts. The pH-stable medium did not affect the in vivo developmental competence of the vitrified-warmed embryos. A farrowing rate of 71.4% (5/7) with 10.4 ± 3.1 piglets born was obtained for the embryos vitrified and warmed in this medium and transferred to selected recipients. This medium will enable the use of simple, safe and standardized protocols for the vitrification and warming of porcine embryos for optimal embryo survival and quality when applied under field conditions. This study opens new possibilities for the widespread use of embryo transfer in pigs.

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ContentsMore than eighteen years have passed since the first derivation of human embryonic stem cells (ESCs), but their clinical use is still met with several challenges, such as ethical concerns regarding the need of human embryos, tissue rejection after transplantation and tumour formation. The generation of human induced pluripotent stem cells (iPSCs) enables the access to patient-derived pluripotent stem cells (PSCs) and opens the door for personalized medicine as tissues/organs can potentially be generated from the same genetic background as the patient recipients, thus avoiding immune rejections or complication of immunosuppression strategies. In this regard, successful replacement, or augmentation, of the function of damaged tissue by patient-derived differentiated stem cells provides a promising cell replacement therapy for many devastating human diseases. Although human iPSCs can proliferate unlimitedly in culture and harbour the potential to generate all cell types in the adult body, currently, the functionality of differentiated cells is limited. An alternative strategy to realize the full potential of human iPSC for regenerative medicine is the in vivo tissue generation in large animal species via interspecies blastocyst complementation. As this technology is still in its infancy and there remains more questions than answers, thus in this review, we mainly focus the discussion on the conceptual framework, the emerging technologies and recent advances involved with interspecies blastocyst complementation, and will refer the readers to other more in-depth reviews on dynamic pluripotent stem cell states, genome editing and interspecies chimeras. Likewise, other emerging alternatives to combat the growing shortage of human organs, such as xenotransplantation or tissue engineering, topics that has been extensively reviewed, will not be covered here.

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