Combining Breeding and Advanced Reproduction Techniques for Regenerative Medicine

Organ regeneration by using pluripotent stem cells is one of the ultimate goals of regenerative medicine. We are currently conducting a study on blastocyst complementation where mechanisms involved in early embryogenesis and fetal development processes is used to generate organs and tissues in vivo in pigs from exogenous pluripotent cells. The strategy involved creating genetically modified pigs lacking a specific organ, and inducing cloned embryos of the organogenesis-disabled pig into a chimeric state, wherein the lacking organ is generated from the exogenous pluripotent cells. We have successfully created pancreatogenesis-disabled pigs through overexpression of the Pdx1-Hes1 transgene and Pdx1 gene knockout. Chimeric pigs were created by blastocyst complementation, in which cloned embryos of the apancreatic pigs were used as host embryos, and cloned embryos of normal pigs were used as donors. The resulting chimeric pigs were confirmed to have formed pancreas from the exogenous cells, i.e. the donor cells. The chimeric pigs demonstareted normal physiological characteristics, including pancreatic functions, and developed into adults with normal reproductive ability. Thus, we showed that the formation of fully functional organs derived from allogenic exogenous cells could be achieved in vivoin apancreatic pigs by using blastocyst complementation technology. The next challenge is to determine whether interspecies blastocyst complementation using the apancreatic pigs as the platform is possible.

     Recent advances in genome editing technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas have also enabled the generation of genetically modified pigs as human disease models. The efficiency of inducing indels by introducing mRNAs of ZFN or TALEN into porcine primary cultured cells ranged from 6 to 98 percent depending on the target gene and the genome editing technology used. The gene knockout cells obtained could support embryonic development after nuclear transfer regardless of monoallelic or biallelic knockout. The production efficiencies of the cloned gene knockout pigs were comparable to those of wild-type and transgenic cloned pigs, indicating that the detrimental effect of gene knockout on embryonic/fetal development was limited. While we have been successfully producing gene knockout pigs using a combination of genome editing technologies and somatic cell cloning, the direct injection of mRNAs of ZFN, TALEN, or CRISPR-Cas into zygotes is also a feasible option.

     Thus, reproductive biology plays an indispensable role in the development of regenerative medicine therapies that are expected to bring about a revolution in human health. From our standpoint at the interface of reproductive biology and regenerative medicine, we have been tackling important issues that are directly related to human health. Here, I present an overview of our latest challenges aiming at making the medicine of the future a reality through translational research using as a platform pigs that have a number of similarities with humans. Future prospects for application of genetically engineered pigs in biomedical researches will be discussed in this paper.