**1.1.2 Human induced pluripotent stem cells**

140 Current Frontiers in Cryobiology

using high cryoprotectant concentrations together with flash-freezing in liquid nitrogen has reported higher cell survival rates (Li et al., 2010b; Reubinoff et al., 2001; Richards et al., 2004). However, there are several disadvantages preventing the widespread use of this technique. First, high concentrations of cryoprotectors, which are cytotoxic above 4ºC, are needed. Second, these procedures are tedious to perform manually. Additionally, as vitrification is mostly performed in open pulled straws, contact between the liquid nitrogen and the cells is unavoidable, which carries the risk of contamination. Finally, and one of the most limiting disadvantages of this technique is that it is clearly unsuited for freezing bulk

During the last decade, several groups have been studying different approaches to improve the above described cryopreservation protocols. In the present work we will review the recent advances in the cryopreservation field trying to point out how a better understanding of the sensitivity of hPSCs to the cryopreservation process will help to develop more

The pioneering work on mESCs, and later advances in culturing techniques that were developed to culture nonhuman primate embryonic stem cell lines eventually led to the first successful generation of hESC lines by Thompson and coworkers and two years later by Reubinoff and coworkers (Evans & Kaufman, 1981; Martin, 1981; Reubinoff et al., 2000; Thomson et al., 1995; Thomson et al., 1996; Thomson et al., 1998). These hESCs were derived from human embryos that were produced by *in vitro* fertilization for clinical purposes. HESC lines were karyotypically normal and maintained the developmental potential to contribute to derivatives of all three germ layers, even after clonal derivation and prolonged undifferentiated proliferation (Amit et al., 2000). Since then, hundreds of stem cell lines have been derived world-wide from morula, later blastocyst stage embryos, fresh and cryopreserved supernumerary embryos, single blastomeres and parthenogenetic embryos (Klimanskaya et al., 2006; Lin et al., 2007; Mai et al., 2007; Revazova et al., 2007; Stojkovic et

HESCs grow in tightly packed colonies and maintain defined borders at the periphery of colonies. High nucleus to cytoplasm ratio and prominent nucleoli are typical features of individual hESCs within colonies. HESCs are also characterized by high telomerase activity and expression of a number of cell surface markers and transcription factors including stage-specific embryonic antigen (SSEA)-4, SSEA-3, TRA antigens, Oct3/4, Nanog and absence of hESCs negative markers such as SSEA-1 (Carpenter et al., 2003; Chambers et al., 2003; Draper et al., 2004; Heins et al., 2004; Nichols et al., 1998). Functional confirmation of the multipotent nature of hESCs is generally achieved by examining their potential to differentiate into all three germ layers (ectoderm, mesoderm and endoderm) both *in vitro* and *in vivo. In vitro*, hESCs are allowed to randomly differentiate as embryoid bodies (EBs), which are aggregates of cells grown in suspension culture, followed by immunocytochemical analysis, or measurement of expression of genes associated with the three germ layers by RT-PCR (Reubinoff et al., 2000). The *in vivo* test for pluripotency of hESCs is normally teratoma formation in immunocompromised mice (Bosma et al., 1983).

quantities of hPSCs.

efficient protocols.

**1.1 Human pluripotent stem cells 1.1.1 Human embryonic stem cells** 

al., 2004; Strelchenko et al., 2004).

An important revolution in the stem cell research field was accomplished when several groups in different studies demonstrated that using a cocktail of four factors, somatic cells could be reprogrammed into iPSCs (Maherali et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). Consequently, it was shown that such cells could be generated from patient-specific cells for a wide variety of diseases (Kiskinis & Eggan, 2010; Raya et al., 2009; Raya et al., 2010) and from a wide variety of somatic cell types (Sun et al., 2010). These cells are morphologically similar to hESCs, express typical hESC-specific cell surface antigens and genes, differentiate into multiple lineages *in vitro*, and form teratomas containing differentiated derivatives of all three primary germ layers when injected into immunocompromised mice. Indeed, these new pluripotent cell lines satisfy all the original criteria proposed for hESCs (Thomson et al., 1998). Nevertheless, some differences have been observed between hESCs and iPSCs (Chin et al., 2009); but it remains unclear whether the small percentage of genes that are differentially expressed between these two types of hPSCs are shared among different lines and whether these differences are biologically significant.

Developing iPSCs into therapeutic reagents faces a number of practical hurdles, including risks associated with cell processing, the difficulty of ensuring the purity and characteristics of the reprogrammed population and the safety and efficacy of reprogrammed cells *in vivo* (Condic & Rao, 2008; Rao & Condic, 2008; Rao & Condic, 2009). Moreover, a case of rejection has been recently described after iPSCs autologous transplantation (Apostolou & Hochedlinger, 2011). Nonetheless, there is cause for considerable optimism that patientspecific iPSC lines will both enhance the study of human diseases and advance these studies toward clinical applications.
