In this study, we demonstrate for the first time that mice can be cloned from intact urine-derived cells without harm to the animal’s body. There are several methods for collecting donor cells from animals noninvasively. For example, cells can be obtained from the skin surface by scratching or from hair follicles. However, these cells are usually dried and have a hardened cell surface compared with living cells. These cells can be used to extract DNA for the purpose of identifying victims or parentage. However, it is difficult to use these cells for NT for technical reasons.

In this study, two or three different types of cells could be identified in the mouse urine samples. These cells could be squamous epithelial cells from the urethra and bladder and renal tubular cells9. Some could not be used because of their large size and hard cell surface. However, the small and round cells with a soft surface could be used as nuclear donors immediately after collection. It was easy to inject their nuclei into enucleated oocytes and the ease of injection of these cells was similar or superior to the injection of cumulus cells and fibroblasts. Moreover, most of the urine-derived cells survived until use.

The ingredients contained in mammalian urine are not optimal for cell survival. The osmolality is high and uric acid and ammonia are toxic. Although mice have the enzyme uricase24, which converts uric acid to the less toxic allantoin, urine is still not an appropriate environment for survival of somatic cells. Therefore, it was thought that even if live cells could be collected from urine8,10, the extreme environment would negatively affect cell survival and nuclear integrity. However, we generated male and female cloned mice from young and old mice and demonstrated their fertility by mating them with each other and producing offspring. This strongly suggests that newly collected fresh and intact urine-derived cells are sufficiently viable to be used as nuclear donors for animal cloning.

In this study, we also tried to establish ntES cell lines from urine-derived cell nuclei. The establishment rate was high (24%) compared with full-term development (1–3%); these values are similar to those reported previously22,23. When the establishment rate was calculated for the urine-derived cells, one cell line was obtained from only 15 urine-derived cells (389 total NT/26 total established cell lines). Therefore, if few donor cells are available, it is better to establish ntES cell lines than to produce cloned mice. Once established, ntES cell lines will divide indefinitely or can be cryopreserved until use. Cloned animals can be produced from ntES cell nuclei via serial NT20,21,25,26. Thus, establishing ntES cell lines provides a better overall chance of cloning animals from endangered species. We are now trying to generate chimeric mice using these ntES cells.

The numbers and concentrations of cells in urine varied between individual mice and some individuals provided only a few cells in urine (Fig. 1C). It is unclear why cell numbers differed between individual mice because all mice were housed in a clean facility (SPF condition) and therefore all could have been considered to be in good health. The number of cells collected for urine from wild animals is expected to vary widely between individuals and to reflect the state of health, such as the presence of urinary tract infection.

One advantage of the NT method is that only a few cells are required as nuclear donors. Although the current success rate of animal cloning is not high, cloning procedures and the success rates are continuing to improve with new methods such as adding histone deacetylase inhibitors to the medium16,18,27. The number of required donor cells will continue to decrease with increased success rate; therefore, in the future, fewer urine-derived cells will be needed to rescue endangered species.

Recently, one cloned cow was generated from urine-derived cells, but clean cells were selected and cultured until a sufficient number of cells was achieved after several passages28. However, in practical terms, it is difficult to collect fresh urine from individual animals in the field, especially from small animals. If urine is collected long after urination, the urine-derived cells may be infected or damaged and it may be difficult to increase the number of cells by in vitro culture. Our previous study demonstrated that cloned mice could be produced even from dead cells of frozen cadavers26. Therefore, even if urine-derived cells are not fresh or are dead because of delayed collection, it may still be possible to produce cloned animals from endangered species. Further research is needed to determine how long urine-derived cells can survive after urination and whether dead cells can be used for NT.

In conclusion, our study suggests that urine may be a good source of cells for NT to rescue endangered species without causing harm to animals.