A limited number of studies have evaluated primary cultures

A limited number of studies have evaluated primary cultures of undifferentiated spermatogonia established from adult mice (Schmidt et al., 2011; Seandel et al., 2007), but the relative SSC content and regenerative integrity over an extended period of in vitro proliferation have not been reported. Indeed, a majority of studies regarding culture of rodent SSCs utilized testis cells isolated from prepubertal or neonatal donors. However, studies of Seandel et al. (2007) reported persistence of SSCs over a 2-year period in cultures established from adult testis cells, although quantification of SSC content at various points throughout the culture period were not provided. Moreover, maintenance of regenerative integrity to re-establish the entire spermatogenic lineage was not assessed. Because parameters associated with cultures from adult mice are reflective of establishing cultures from men, assessing the efficiency by which they can be established and maintenance of SSC regenerative integrity is imperative. In the present study, we found a negative association between time of maintenance and regenerative integrity of SSCs in primary cultures of undifferentiated spermatogonia established from cells isolated from testes of pup and adult mice that were maintained in conventional conditions. To address the deficiency, we sought to refine culture conditions based on our understanding of the procollagen c proteinase of SSCs. Our previous studies revealed that expression of key enzymes that regulate glycolysis are elevated in SSCs of primary undifferentiated spermatogonial cultures (Chan et al., 2014). Thus, we examined the impacts of modifying culture conditions to favor utilization of glycolysis as the primary bioenergetics process. When maintained in glycolysis-optimized (GO) conditions, the capacity of SSCs to regenerate complete colonies of spermatogenesis was significantly improved compared with conventional conditions for both pup and adult primary cultures. These findings suggest that a distinguishing characteristic of SSCs is priming to thrive in conditions favoring glycolytic activity, and matching culture conditions to the bioenergetics of the cells is important for maintaining functional integrity.
Results
Discussion Expansion of SSC numbers in vitro while maintaining the integrity to regenerate complete colonies of spermatogenesis is paramount for application of transplantation methodology as a means to preserve and re-establish the fertility of male cancer patients. Because the proliferation rate of SSCs is relatively slow, ∼6 days (Kubota et al., 2004b), a period of long-term maintenance in culture is needed to provide a sufficient number of cells for transplantation if the intent is to yield enough sperm for a pregnancy to be achieved. In addition, long-term culture of SSCs provides a valuable platform to study the mechanisms controlling their fate decisions. Several studies have reported long-term maintenance of putative human SSCs (Sadri-Ardekani et al., 2009, 2011). Although the outcomes were promising, unequivocal determination of SSC presence in human testicular cell cultures is challenging because assessment is restricted to expression of molecular markers whose SSC specificity is undefined and the appearance of human cells in testes of immunodeficient recipient mice after xenotransplantation. Although some of the markers used are expressed by somatic cells, others are expressed by spermatogonia but are not SSC specific. Also, xenotransplantation does not result in regeneration of the spermatogenic lineage; thus, whether bona fide SSCs were present in the human cultures has not been truly demonstrated. Two major conventional methodologies for long-term culture of mouse undifferentiated spermatogonial populations that contain bona fide SSCs were described over 10 years ago. Studies of Kanatsu-Shinohara et al. (2003) developed methodology that includes a complex medium formulation based on the commercial supplement StemPro and co-culture with MEF feeder cells, which supports long-term maintenance and growth of a primary undifferentiated germ cell population derived from testes of neonatal (P0) mice. Although an exciting breakthrough, these cultures were derived from prospermatogonia that are present at P0, which are precursors to undifferentiated spermatogonia. Also, the conditions were developed for mice with a DBA/2 genetic background in which adult stem cells are known to possess enhanced capacity for proliferation and survival compared with other strains of mice (Muller-Sieburg and Riblet, 1996). The relevance of these conditions to actual undifferentiated spermatogonia including SSCs in either prepubertal pups or adults and other strains of mice has remained relatively undefined. Subsequently, Kubota et al. (2004b) developed a method that includes a defined medium formulation and co-culture with STO feeder cells that supports long-term maintenance and growth of a primary undifferentiated spermatogonial population derived from P6–P8 mice of several genetic backgrounds including DBA/2 and C57BL/6. In contrast to claims by Kanatsu-Shinohara et al. (2016) that derivation of cultures is notoriously difficult with mice of a C57BL/6 background, a multitude of studies have utilized the method of Kubota et al. (2004b) to derive long-term cultures of cells from P6–P8 mice with this genetic background and utilize them successfully for experimentation of the SSC pool (Chan et al., 2014; Kaucher et al., 2012; Lovelace et al., 2016; Oatley et al., 2006, 2007, 2009, 2010, 2011; Wu et al., 2010; Yang et al., 2013a, 2013b, 2013c). It is important to note that for both of the conventional methods, only a minor portion of the germ cell population possesses the capacity to regenerate spermatogenesis following transplantation into recipient testes. Hence, the cultures cannot be considered pure SSCs or germline stem (GS) cells. In actuality, the cultures are a primary population of undifferentiated spermatogonia if derived from prepubertal pups and prospermatogonia if derived from P0 neonates.