However before application in a clinical

However before application in a clinical context, a number of issues still need to be considered. Indeed, SSC transplantation in cancer patients requires techniques to exclude cancer cell contamination, because implantation of as few as 20 leukemic nrf2 inhibitor can result in cancer relapse (Jahnukainen et al., 2001), and approximately 21% of boys with leukemia show intratesticular infiltration by malignant cells (Kim et al., 1981; Kim et al., 1986). Several cell sorting methods have been evaluated for selective isolation of human SSCs, but none has so far managed to achieve complete elimination of all cancer cells without pretreatment of cells to be sorted (Fujita et al., 2006; Geens et al., 2007; Dovey et al., 2013). However, using specific culture conditions, Sadri-Ardekani and colleagues were able to eliminate leukemic cells when co-cultured together with testicular cells. Cancer cells were undetectable by PCR after 26days of culture, even if present at a high initial concentration (40%), suggesting that this testicular cell culture system allows propagation of SSCs while removing contaminating cancer cells (Sadri-Ardekani et al., 2014). Another important concern is the genetic integrity of propagated SSCs and their potential impact on offspring when transplanted. Indeed, in mice, the SSC karyotype was normal after 24months of culture (Kanatsu-Shinohara et al., 2005), but in human, although the SSCs remained euploid, there were changes in their methylation status after 50days of culture (Nickkholgh et al., 2014a). Further studies are therefore required to investigate possible implications on the functionality of propagated SSCs, and hence on derived mature germ cells.
Towards obtaining in vitro-matured spermatozoa The first report describing in vitro completion of meiosis in vitro (Weiss et al., 1997) via co-culture of rat Sertoli cells and pachytene spermatocytes, was published in 1997 and fertile offspring were obtained in mice using sperm produced in organotypic culture of fresh ITT in 2011 (Sato et al., 2011). However, in humans, the completion of the spermatogenic process could not be reproduced in vitro, despite development of different culture systems, such as co-culture with Vero cells (Cremades et al., 2001; Sousa et al., 2002), isolated cell culture with growth factor supplementation (Yang et al., 2014), 3D culture (Lee et al., 2007) and organotypic culture (Roulet et al., 2006). Results achieved with Vero cells, even if promising, will not be discussed here, as their animal origin precludes clinical application. Organotypic culture has the advantage of preserving interactions inside the SSC niche and with the interstitial compartment, potentially favoring the complex cellular relationships in the original tissue architecture. So far, the focus has been mainly on the culture medium. While use of fetal bovine serum (AlbuMAX, a lipid-rich albumin fraction of bovine serum) and transferrin appeared to be key factors for IVM of mouse ITT (Sato et al., 2011), such animal-derived media cannot be used in humans. The pioneer of human models for IVM of testicular tissue was Steinberger (1967) who applied tritiated thymidine to fragments of adult testes to demonstrate partial spermatogenesis from the preleptotene to the pachytene stage using basic culture medium (Steinberger, 1967). Ghatnekar confirmed Steinberger\'s results, achieving differentiation of leptotene spermatocytes up to telophase II in organ culture of adult testicular tissue using a culture medium enriched in FSH and luteinizing hormone (Ghatnekar et al., 1974). Roulet also evidenced differentiation of preleptotene spermatocytes into pachytene spermatocytes, but observed considerable loss of germ cells (GCs) despite addition of gonadotropins after 16days of organotypic culture of adult testicular tissue (Roulet et al., 2006). Apart from the lack of complete spermatogenesis and progressive loss of GCs, a further issue emerging from studies is the difficulty of assessing the actual steps accomplished in culture. This is due to use of adult tissue, namely men presenting with normal spermatogenesis (Roulet et al., 2006), where haploid cells may already be present in the tissue before culture. Some study results are even more questionable. Indeed, Tesarik et al. (Tesarik et al., 1999), observed round spermatids after only 48h, while the duration of meiosis is much longer according to human physiology.