Research shows major differences between mouse and primates in the behavior of early embryos toward introduced ESCs.
Experiments leading to the birth of the world’s first chimeric rhesus monkeys have demonstrated that mouse and primate early embryos have very different capacities to accept pluripotent cells and develop into true chimeras. The three male infants, twins Roku and Hex, and a singleton called Chimero, were born after researchers at the Oregon National Primate Research Center and Oregon Health & Science University generated aggregates of multiple four-cell embryos, and implanted the resulting blastocysts into surrogate mothers.
Describing their achievement in Cell, Shoukhrat Mitalipov, Ph.D., and colleageus describe work leading up to the birth of the rhesus babies that shows how, in contrast with mouse cells, rhesus monkey embryonic stem cells (ESCs) and isolated inner cell masses (ICMs) fail to incorporate into host embryos and develop into chimeras. Whereas introduducing cultured ESCs into mouse embryos is a standard method for genering chimeric mice, this method failed every time in primates. The Oregon team reports in a paper titled “Generation of Chimeric Rhesus Monkeys.”
When mouse ESCs that have been propagated in vitro are introduced into mouse blastocyts, the transplanted cells engraft into ICM and participate fully in the development of chimeric fetuses and offspring. In fact, a range of donor cell types have been found to support mouse chimera production, including teratocarcinoma cells, embryonic germ cells, and induced pluripotent stem cells (iPSCs) and pluripotent cells generated by somatic cell nuclear transfer. This ability of introduced mouse stem cells to contribute to chimeric fetal development has become the “ultimate test” for pluripotency, the authors write. However, what isn’t yet known is whether primate pluripotent cells retain equivalent features.
The Oregon team evaluated the ability of rhesus monkey ESCs to contribute to chimeric fetusus after injection into in vitro fertilization-derived host blastocyts. Either GFP-tagged, or untagged (i.e., nontransgenic) ESCs were injected next to the inner cell mass of 26 rhesus monkey blastocysts, and the blastocysts then immediately transplanted into seven synchronized surrogates. Of these, four became pregnant (one carrying quadruplets and three carrying singletons).
Interestingly, mitochondrial DNA parentage analysis, direct GFP fluorescence analysis, and microsatellite parentage analysis of genomic DNA failed to identify any contribution of the transplanted ESCs to any tissues of the resulting fetuses at mid-gestation. To investigate whether this failure was due to the limited developmental potential of ESCs or to an inability of host blastocysts to incorporate foreign embryonic cells, the researchers injected freshly isolated whole ICMs into host blastocysts from unrelated monkeys, and these blastocysts were then implanted into surrogates. The three resulting fetuses were all recovered at mid-gestation and analyized for any contribution of transplanted ICMs to development.
One of the surrogates had developed a monochorionic di-amniotic twin pregnancy, and in this case the fetuses were, unexpectedly, different genders. Analyses of these indicated that one originated from the host blastocyst, whereas the second derived from the injected ICM. There was no chimerism in any tissues apart from the livers and spleens of both twins, which the authors note may have resulted from the exchange of blood and hematopoietic progenitors through placental perfusion. The single male fetus developing in the other surrogate mother appeared to be derived solely from the injected ICM, whereas the placental component was female, and mainly contributed by the host blastocyst.
“These results demonstrate that contrary to the mouse and some other species, monkey blastocysts do not readily incorporate ESCs or foreign ICMs and form embryo proper chimeras,” the team writes. “However, transplanted ICMs were capable of forming separate viable fetuses while sharing the trophectodermal compartment of the host embryo.”
Moving back a developmental stage, the team then investigated whether the totipotent blastomeres of cleaving embryos would be able to incorporate foreign blastomeres and form chimeras. They initially tried to generate chimeric embryos by replacing two blastomeres in the four cell-stage embryo with two blastomeres from different embryo at the same developmental stage. Encouragingly, a limited number of these mixed four-cell embryos did generate chimeric blastocysts, so the team then carried out further studies in which whole embryos were aggregated to try and increase the yield of chimeric blastocysts and potentially offspring.
Using between three and six individual four-cell stage embryos, the investigators generated 29 aggregates and successfully cultured all to the blastocyst stage. The vast majority exhibited more than double the normal cell counts, indicating that aggregation had been successful. Fourteen of these were transplanted into five recipient females. All the surrogates became pregnant. This in itself was surprising, because pregnancy and implantation rates in nonmanipulated rhesus embryos generally don’t exceed 36% and 17%, respectively, the team notes. “High pregnancy and implantation results observed with chimeric blastocysts suggest that higher cell numbers in embryos are critical for pregnancy initiation.”
Three of the pregnancies were subsequently terminated and seven fetuses recovered for genetic analysis. All were found to be true chimeras and displayed chimerism in all the sampled organs and tissues. In some cases at least three different embryos had contributed to chimeric tissues.
The remaining pregnancies were allowed to progress to term: one of the two mothers gave birth to healthy male twins (Roku and Hex), and the other to a healthy male singleton (Chimero). All three rhesus babies were true chimeras. “To our knowledge, these infants are the world’s first primate chimeras,” the authors state.”
Some of the contributing embryos used to generate the chimeric infants were female, and despite the fact that all three were phenotypically male, detailed cytogenetic analyses confirmed that in Roku’s case at least, the infant’s blood contained both male and female cells, and molecular cytogenetic studies indicated that about 4% of Roku’s cells analyzed were XX rather than XY.
Because the four-cell embryos also appeared to also accept blastomeres from other embryos, the researchers investigated whether four-cell embryos might accept injected ESCs and develop into chimeras. GFP-expressing monkey ESCs were injected under the zona pellucida and placed between blastomeres of four-cell embryos. The resulting aggregates were cultured to the blastocyst stage and six GFP-positive embryos were implanted into two recipient mothers. One of these became pregnant with a singleton. Again, however, analysis of the fetus at midgestation found no contribution of ESCs to tested tissue and organs.
To try and find out why, the authors injected GFP-expressing ESCs into four-cell embryos and examined the resulting blastocysts. The results of this indicated that although the cleaving host embryos can incorporate ESCs, they can’t support undifferentiated growth of ESCs, and over the 4–5 day period during which the injected embryos develop into blastocysts, the ESCs undergo differentiation and lose pluripotency. “This phenomenon is likely to preclude contribution of ESCs to the fetal tissues and organs,” they remark.
The upshot is that totipotent, cleaving monkey embryos can’t serve as a host for testing pluripotency of ESCs, the team sates. “Unlike mouse, where two- to eight-cell stage embryos engraft ESCs and form chimeras, cleaving primate embryos do not seem to provide a niche for undifferentiated maintenance of ESCs until the host ICM is formed.”
Little is currently known about how closely mouse embryo development reflects primate embryo development, they continue. “Our study presents an indication of the similarities and differences between mouse and primate preimplantation embryo development and offers an important experimental model to investigate lineage commitment and interactions.”
