**4.3 Stem cell preservation**

332 Current Frontiers in Cryobiology

15,000 year-old wooly mammoth (*Mammuthus primigenius*) to a mouse (Kato et al., 2009). While this technique holds much promise for the resurrection of extinct species and saving those on their way there (Loi et al., 2011b), with the exception of a few sporadic instances, all these attempts at ISCNT did not result in live offspring. Cryopreservation of reproductive tissue or any other viable body tissue or, alternatively, of *in vitro* grown cell cultures is routinely done in many places around the world and enough cells survive the process to be used in SCNT. Furthermore, obtaining tissue samples is usually much simpler than collecting gametes or embryos, so a larger and more diverse collection can be accumulated. While SCNT has the advantage that no genetic drift takes place because recombination does not occur, when considering SCNT for wildlife species preservation, several important issues should be taken into consideration. First, as mentioned above, suitable enucleated oocytes are required. The availability of such oocytes and the ability to access them should thus be part of the program (Loi et al., 2011b). If conspecific oocytes are not available, the issues of mitochondrial inheritance and nucleus-cytoplasmic incompatibility become a problem and ways to overcome these should be sought for. When the donor and recipient are close enough, some of the donor mitochondria get transferred as well (Gómez et al., 2009; Srirattana et al., 2011). As was demonstrated for the famous sheep, Dolly, the telomere is shorter following SCNT (Shiels et al., 1999). Interestingly, it was recently shown that cloned cows with short telomeres produce normal and healthy offspring with normal telomere length following artificial insemination with sperm from normal bulls (Miyashita et al., 2011). This study suggests that cloning does not interfere with the eventual function of the germ line. Cloned offspring, however, are known to show elevated prevalence of developmental abnormalities and high mortality rate, issues that should be kept in mind when initiating a cloning program (e.g. Lanza et al., 2000). One should also keep in mind that the spermatozoa carry more than just genetic material. They come with a whole load of epigenetic factors important for proper embryonic development (Yamauchi et al., 2011). These are missing when SCNT is performed and might be one of the causes behind the relatively low efficiency of the process. As with cryopreservation of other cells and tissues, storage space and costs and environmental impact are major issue pertaining to liquid nitrogen storage so a cheaper alternative would be very

In tissue banking, as in the banking of germ cells and embryos, storage and maintenance costs are always an issue because of the properties of liquid nitrogen. Seeds of plants, having low water content are relatively easy to preserve at high subzero temperatures (-20 to -30ºC). With water content of about 80%, preservation of gametes and embryos in the animal kingdom is complicated and species-specific. The use of large quantities of liquid nitrogen for cryopreservation and storage also has its toll on the environment, as the production of liquid nitrogen is energy-intensive, resulting in the release of large quantities of carbon dioxide. An alternative to cryopreservation of somatic cells, then, can be to dry them and store the dry cells at room temperature. While, as was discussed earlier, sperm drying has been achieved in a number of species, the parallel in females, namely oocyte drying, is yet to be demonstrated. Somatic cell drying is thus the way to go when long-term storage for females or of the entire genetic complement is desired. In this respect, the use of sheep freeze-dried somatic cells for SCNT was recently demonstrated (Loi et al., 2008a; Loi et al., 2008b). In their report, utilizing the directional freezing technology, freeze-dried

attractive for long-term conservation purposes.

**4.2 Somatic cell drying for SCNT** 

Embryos can be a source for primordial germ cells (PGC) which, as was shown in the zebrafish, can be vitrified, warmed and then transplanted into sterilized recipient blastulae to differentiate into males and females that produced gametes carrying the genetic material of the transplanted PGC donor (Higaki et al., 2010). Such PGC can be transplanted, along with gonadal somatic cells, and develop into normal male or female gonadal tissue with normal spermatogenesis or oogenesis. Both mouse round spermatids and GV oocytes derived from such tissues were able to direct embryonic development to term following ICSI (Matoba & Ogura, 2010). In a recent study on felids (Silva et al., 2011) it was shown that such germ line stem cells can be transplanted to the gonads of a different species and still develop normal early stage gametes. In that study, ocelot (*Leopardus pardalis*) spermatogonial stem cells were transplanted into domestic cat testis and thirteen weeks later ocelot spermatozoa were retrieved from the cat's epididymis.

Going even earlier in the development timeline, embryos can be a source for stem cells. Embryonic stem cells, being pluripotent, can differentiate *in vivo* or *in vitro* into germ cells. They can also be used for nuclear transfer. So, they, too, can be considered an optional venue. In a study on mice, transplanted embryonic stem cells were able to form testicular tissue structures and direct spermatogenesis (Toyooka et al., 2003). These cells, which can be isolated from embryos, can also be cryopreserved (Thomson et al., 1998; Toyooka et al., 2003) or vitrified (Reubinoff et al., 2001; He et al., 2008). Such stem cells can also be derived from embryos generated by nuclear transfer of freeze-dried cells (Ono et al., 2008). Embryonic stem cells can also be derived from isolated blastomeres, and blastomers can also be cryopreserved individually by inserting them into emptied zona pellucida and then vitrifying them (Escriba et al., 2010). If embryonic stem cells are not available, somatic cells can be induced to become embryonic stem cells-like (Takahashi & Yamanaka, 2006), also known as induced pluripotent stem cells or iPS cells (for recent review see: Cox & Rizzino, 2010). Being pluripotent in nature, they are also germ line competent (Okita et al., 2007) and as such can give rise to germ cells of both male and female.

The fantastic options mentioned above are theoretical and speculative in nature when it comes to wildlife preservation as currently these techniques are in their infancy and were adapted thus far only to laboratory animals, and even in these the unknown is still vast.
