Tetraploid complementation assay

The tetraploid complementation assay is a technique in biology in which cells of two mammalian embryos are combined to form a new embryo. ^[1] It is used to construct genetically modified organisms, to study the consequences of certain mutations on embryonal development, and in the study of pluripotent stem cells. The first demonstration that induced pluripotent stem cells (iPSCs) could generate viable mice through tetraploid complementation was reported in 2009, providing strong functional evidence that iPSCs can be equivalent to embryonic stem cells in developmental potential. ^[2]

Procedure

Normal mammalian somatic cells are diploid: each chromosome (and thus every gene) is present in duplicate (excluding genes from X chromosome absent in Y chromosome). The assay starts with producing a tetraploid cell in which every chromosome exists fourfold. This is done by taking an embryo at the two-cell stage and fusing the two cells by applying an electrical current. The resulting tetraploid cell will continue to divide, and all daughter cells will also be tetraploid.

Such a tetraploid embryo can develop normally to the blastocyst stage and will implant in the wall of the uterus. The tetraploid cells can form the extra-embryonic tissue (placenta, etc.); however, a proper fetus will rarely develop.

In the tetraploid complementation assay, one now combines such a tetraploid embryo (either at the morula or blastocyst stage) with normal diploid embryonic stem cells (ES) from a different organism. The embryo will then develop normally; the fetus is exclusively derived from the ES cell, while the extra-embryonic tissues are exclusively derived from the tetraploid cells.

Applications

Tetraploid complementation is often described as one of the most stringent functional tests of pluripotency in mammals, because it can show whether pluripotent stem cells are able to generate the embryo proper when supported by tetraploid extraembryonic tissues. ^[3]

• Foreign genes or mutations can be introduced into ES cells rather easily, and these ES cells can then be grown into whole animals using the tetraploid complementation assay.
• By introducing targeted mutations into the tetraploid cells and/or into the ES cells, one can study which genes are important for fetal development and which ones are important for development of the extra-embryonic tissues.
• The tetraploid complementation assay is also used to test whether induced pluripotent stem cells (stem cells artificially produced from differentiated cells, e.g. from skin cells) are as competent as normal embryonal stem cells. If a viable animal can be produced from an induced pluripotent stem cell using the tetraploid complementation assay, then the induced stem cells are deemed equivalent to embryonal stem cells. This was first shown in 2009. ^[4] In humans, tetraploid complementation is not used to assess induced pluripotent stem cell potential due to ethical and legal constraints on characterizing human stem cell lineages using chimeric embryo assays like tetraploid complementation. ^[3]
• After ES cells editing, the tetraploid complementation assay can be used to generate genetically modified mouse model (directly produce a viable animal without pronuclear or Blastocyst microinjection. ^[5]

Although the assay was developed and is most widely used in mice, tetraploid complementation approaches have also been applied in other mammals, including rats, where embryonic stem cells have been used to generate viable animals via tetraploid complementation, demonstrating the broader utility of the assay. ^[6]

Limitations and ethical considerations

Tetraploid complementation involves generation of chimeric embryos and requires in utero gestation, which poses ethical issues that preclude its use in human pluripotent stem cell research. International stem cell research guidelines emphasize ethical oversight and prohibit manipulation of human embryos using techniques such as tetraploid complementation. ^[7]

References

1. Mouse embryonic chimeras: tools for studying mammalian development, Development 130, 6155-6163 (2003)
2. Hamanaka, Shinya (2009). "Generation of germline-competent induced pluripotent stem cells". Nature. 461: 93–97. doi:10.1038/nature08304.
3. "Evaluating Strategies to Assess the Differentiation Potential of Pluripotent Stem Cells". Stem Cell Reviews and Reports. 2025. Retrieved 6 February 2026.
4. Lan Kang, Jianle Wang, Yu Zhang, Zhaohui Kou, Shaorong Gao. 2009. iPS cells can support full-term development of tetraploid blastocyst-complemented embryos. Cell Stem Cell. doi : 10.1016/j.stem.2009.07.001 PMID   19631602
5. Liu, F L, Wu K, Sun J, Duan Z, Quan X, Kuang J, Chu S, Pang W, Gao H, Xu L, Li Y C, Zhang H L, Wang X H, Luo R H, Feng X L, Scholer H R, Chen X, Pei D, Wu G*, Zheng Y T*, Chen J*. Rapid generation of ACE2 humanized inbred mouse model for COVID-19 with tetraploid complementation[J]. Natl Sci Rev, 2021, 8(2): nwaa285.
6. Li, T. D. (2017). "Rat embryonic stem cells produce fertile offspring through tetraploid complementation". Proceedings of the National Academy of Sciences. doi:10.1073/pnas.1708710114.
7. "ISSCR Guidelines for Stem Cell Research". International Society for Stem Cell Research. Retrieved 6 February 2026.