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Generation of Chimeric Rhesus Monkeys

From Cell
By Stuart P. Atkinson

A recent research article in Cell from the laboratory of Shoukhrat Mitalipov at the Oregon National Primate Research Center at the Oregon Health & Science University has reported the generation of the first chimaeras from a non-human primate (Tachibana et al). In mouse, the ability to contribute to chimeric animals upon re-introduction into host embryos is the key feature of totipotent and pluripotent cells, and while chimaeric animals have been produced in other mammals (rats, rabbits, sheep and cattle) this had not been extended to non-human primates. Further as relatively little is known about human and non-human primate embryo development and lineage specification and how closely the mouse development reflects primates, research such as this promises to assess the usefulness of mouse models and mouse embryonic stem cells (mESCs) to human embryonic stem cell (hESC) biologyInitial analysis of rhesus monkey ESCs (rmESCs) ability to contribute to chimeric foetuses through in vitro fertilisation-derived host blastocysts demonstrated that out of 26 injected blastocysts transplanted into seven synchronized recipients four females became pregnant—one carrying quadruplets and three carrying singletons with the remaining three recipients containing gestational sacs without foetuses.   Pregnancies were terminated at mid gestation to allow detailed analysis which found that there was no evidence that rmESCs contributed to the foetuses, suggesting a limited developmental potential of the rmESCs or the host blastocysts inability to incorporate the rmESCs.   To investigate these possibilities, non-cultured pluripotent cells (from whole inner cell mass, ICM) were injected into host blastocysts and of the 11 recipients, two females were confirmed pregnant, with one being a mono-chorionic twin pregnancy.   Interestingly, it was found that one foetus originated from the host blastocyst, whereas the second foetus was derived from the injected ICMs and chimerism was detected only in liver and spleens of both foetuses, perhaps mediated through the exchange of blood and hematopoietic progenitors through placental perfusions.   The placental sample contained a mixture of several extra-embryonic membranes and was contributed by both the host embryo and injected ICM.   The foetus associated with the singleton pregnancy was male and originated solely from the injected ICM, whereas the placental (trophectoderm) component was female and mainly contributed by the host blastocyst.

Initial analysis of rhesus monkey ESCs (rmESCs) ability to contribute to chimeric foetuses through in vitro fertilisation-derived host blastocysts demonstrated that out of 26 injected blastocysts transplanted into seven synchronized recipients four females became pregnant—one carrying quadruplets and three carrying singletons with the remaining three recipients containing gestational sacs without foetuses. Pregnancies were terminated at mid gestation to allow detailed analysis which found that there was no evidence that rmESCs contributed to the foetuses, suggesting a limited developmental potential of the rmESCs or the host blastocysts inability to incorporate the rmESCs. To investigate these possibilities, non-cultured pluripotent cells (from whole inner cell mass, ICM) were injected into host blastocysts and of the 11 recipients, two females were confirmed pregnant, with one being a mono-chorionic twin pregnancy. Interestingly, it was found that one foetus originated from the host blastocyst, whereas the second foetus was derived from the injected ICMs and chimerism was detected only in liver and spleens of both foetuses, perhaps mediated through the exchange of blood and hematopoietic progenitors through placental perfusions. The placental sample contained a mixture of several extra-embryonic membranes and was contributed by both the host embryo and injected ICM.   The foetus associated with the singleton pregnancy was male and originated solely from the injected ICM, whereas the placental (trophectoderm) component was female and mainly contributed by the host blastocyst.

Next, the ability of totipotent blastomeres (4 cell stage) to form chimaeras was investigated, based on the research showing that an isolated single blastomere from this stage embryo can implant and develop into a viable rhesus offspring (Chan et al). 29 chimeric 4-cell embryos, of which 19 reached blastomere stage, were analysed, with no embryo developing normally. This led the researchers to attempt the aggregation of three or more whole cleaving embryos to allow better contact between blastomeres and ensure that at least two of these embryos would develop to blastocysts and contribute to chimeric ICMs. Amazingly, this led to 29 out of 29 aggregates developing to blastocysts and 26 showing indications of successful aggregation. 14 chimeric blastocysts were transplanted into 5 recipients, with all 5 falling pregnant including two with singletons, two with twins, and one female carrying quadruplets, a remarkably high pregnancy outcome compared to other studies (Wolf et al), so suggesting that higher cell numbers in embryos are critical for pregnancy initiation. All foetuses analysed were chimeric (in all organs sampled) and of normal size and had no obvious defects or congenital abnormalities.   Detailed analysis found that in some chimeras at least three separate genotypes (and therefore embryos) contributed and these were of different genders.   Two recipients carried the pregnancies to term, giving twins (Roku and Hex) and a singleton (Chimero) that were outwardly male, had no obvious congenital abnormalities and were all chimeric, claimed by the researchers to be the world’s first primate chimeras.   Blood samples from Roku contained both male and female cells (4% of cells were XX) demonstrating that they are also sex chimeras.

This better ability of the early stage multiple 4-cell aggregates to form chimeras over the cells of the later stage ICM may be due differences in the level of differentiation status and hence lineage segregation. To analyse this, whole blastocysts or isolated ICMs were immunolabeled for a marker of the epiblast (Nanog) and primitive endoderm (Gata6), and results indicated the presence of spatially segregated Gata6+ and Nanog+ cells, which may lie at the root of the inability of primate ICMs in pre-implantation blastocysts to incorporate foreign pluripotent cells. Next, the ability of rmESCs to incorporate into four-cell embryos and from chimeras was analysed. Resultant aggregated blastocysts where then transferred into hosts, with one singleton pregnancy occurring which did not however show any signs of chimerism, perhaps, as the authors suggests, as the rmESCs are pluripotent rather than totipotent and may prematurely differentiate prior to blastocyst formation. This was confirmed through analysis of GFP+ ESCs injected into 4-cell embryos which demonstrated that while the majority of blastocysts contained embedded rmESCs within trophectoderm or ICM, cells of the ICM were Nanog-, suggesting that these cells are no longer pluripotent, which is likely to preclude contribution of rmESCs to the fetal tissues and organs.

Overall, the paper suggests that rmESCs cannot contribute to chimeras as they represent a more epiblast-like stem cell and are unlike mESCs and have clear implications for the developmental potential of derived stem cells. Primate stem cells (e.g. human ESCs (hESCs)) are comparable to mouse epiblast stem cells which are derived from post-implantation embryos and are developmentally advanced relative to naive mESCs (Nichols and Smith). This suggests that the extrapolation of data derived from mESCs may be inadequate for the advancement of primate ESC biology and suggests the further analysis of hESCs derived from earlier stage blastocysts, such as blastomere-derived hESCs, or from hESCs-derived from ICM under low-oxygen conditions (Lengner et al), which are suggested to exist in a naïve state akin exhibiting an mESC-like phenotype.

 

References

Chan, A.W. et al. (2000).
Clonal propagation of primate offspring by embryo splitting.
Science 287, 317–319.

Mitalipova, M. et al (2010)
Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations.
Cell. 141(5), 872-83.

Nichols, J. and Smith, A. (2011).
The origin and identity of embryonic stem cells.
Development. 138(1), 3-8.

Tachibana, M. et al (2012).
Generation of chimeric rhesus monkeys.
Cell 148, 285-95.

Wolf, D.P. et al (2004).
Use of assisted reproductive technologies in the propagation of rhesus macaque offspring.
Biol. Reprod. 71, 486–493.

See Also:

Chimeric Primates: Embryonic Stem Cells Need Not Apply (Cell, Volume 148, Issue 1, 19-21, 05 January 2012)