Interspecies Chimerism with Mammalian Pluripotent Stem Cells

Abstract

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.

Interspecies chimeras for human stem cell research

“…So, although mouse is the most accessible animal in terms of obtaining embryos, when analyzing human cells other hosts more closely related to humans should be considered. In support of this, Wu et al (2017) reported successful chimera formation using human primed PSCs transplanted into porcine embryos -albeit at a very low rate and degree of chimerism -whereas chimeras were not obtained when rodent naïve PSCs were transplanted. These results highlight the importance of genetic/evolutionary distance when generating interspecies chimeras.…”

“…However, in both cases, the frequency of chimeric embryos among all injected embryos was remarkably low, and the degree of chimerism -the contribution of the PSC-derived cells to the chimera -was far below the level found in mouse-rat interspecies chimera (Kobayashi et al, 2010;Isotani et al, 2011;Yamaguchi et al, 2017;Wu et al, 2017). Other groups have reported that engrafted human naïve-like PSCs disappear after implantation, or in some cases survive but contribute only to the extraembryonic region (Takashima et al, 2014;Masaki et al, 2015).…”

“…Thus, it will be important to carefully characterize the degree of chimerism by human cells in gametes and the central nervous system at the immature fetal stage. Judging from the results published so far for human-animal interspecies chimeras (Masaki et al, 2015;Wu et al, 2017), it is unlikely that chimerism in the central nervous system or in gametes is high enough to cause concern and, in the case of the latter, current restrictions prohibit the breeding of interspecies chimeras. Nonetheless, if the contribution to these organs reaches a problematic level, then it might be necessary to adopt ʻtargeted organ generation' approaches, for example by using genetic methods to restrict developmental potency or by transplantation of apoptosis-resistant progenitor cells with a more restricted and defined cell fate (Masaki et al, 2016).…”

“…However, whether the cells might show an even higher rate of chimerism with porcine embryos was not tested. It has also been shown that human PSC-derived cells in interspecies chimeras can differentiate into multiple lineages (Mascetti and Pedersen, 2016;Wu et al, 2015Wu et al, , 2017Yang et al, 2017), but whether their differentiation was autonomous or due to cooperative morphogenesis with host tissue is controversial. It remains to be seen whether organs and tissues can be cooperatively formed between host cells and transplanted human cells in these interspecies chimeras.…”

Chimeras intended for human gamete production: an ethical alternative?

“…One way in which this can be achieved is through interspecies blastocyst complementation. In this technique, a non-human animal blastocyst is obtained from a mutant strain in which 'a gene critical for the development of a particular lineage is disabled' (Wu et al, 2016(Wu et al, , 2017. Afterwards, this blastocyst is complemented with human stem cells, which will compensate for the existing niche.…”