In the current study we devised

In the current study, we devised simple refinements to the conventional culture condition of Kubota et al. (2004b) that promotes utilization of glycolysis, which we have termed GO (glycolysis-optimized) conditions. To achieve this, we removed all supplementation of lipids and free fatty acids from the medium and reduced the O2 tension to 10%. In these conditions, we found that the integrity of long-term cultured SSCs from both pup and adult mice to regenerate complete colonies of spermatogenesis was significantly improved. Considering that reduced O2 tension is known to enhance the maintenance of somatic stem cell populations in vitro (Csete, 2005; Ishikawa and Ito, 1988; Maeda et al., 1986), it is possible that the reduced O2 level of the GO condition alone was the major influential factor in improving SSC regenerative capacity. However, omission of lipids in addition to the reduced O2 tension did not negatively affect maintenance of the SSC pool during long-term culture for either pup or adult cells. Thus, we recommend that both these modifications be employed in future studies utilizing primary cultures of mouse SSCs and anticipate that they will serve as the basis for developing culture conditions for SSCs from higher-order mammals. Indeed, we discovered in a previous study that long-term culture of cattle undifferentiated spermatogonia is enhanced in an LJI308 of reduced O2 tension (Oatley et al., 2016). In addition, the findings of the current study highlight the importance of matching culture conditions to the bioenergetics of SSCs during long-term in vitro maintenance and provide the impetus for future studies to further refine the conditions to promote utilization of glycolysis.
Experimental Procedures
Author Contributions
Acknowledgments We thank members of the J.M.O. laboratory for valuable input and discussion, and Dr. F. Chan for assistance in data collection. This research was supported by grant HD061665 awarded to J.M.O. from the NICHD.
Introduction Human embryonic stem cells (hESCs) are pluripotent cells having a unique capacity to self-renew and differentiate into all specialized cell types found in somatic tissues (Thomson et al., 1998). Similar properties are gained by human induced pluripotent stem cells (hiPSCs) reprogrammed from cells found in adult tissues (Yu et al., 2007; Takahashi et al., 2007). The core factors known to be crucial for maintenance and control of pluripotency include POU5F1, SOX2, and NANOG (Boyer et al., 2005; Chambers and Tomlinson, 2009). These transcription factors operate in activation or repression of genes important for pluripotency and differentiation, and form an autoregulatory loop to positively regulate their own expression. In addition, POU5F1 and SOX2, in combination with either KLF4 and CMYC or NANOG and LIN28, were used in the first studies reprogramming adult cells back to the pluripotent state, highlighting the importance of these factors in the regulation of pluripotency (Yu et al., 2007; Takahashi et al., 2007). Moreover, epigenetic regulation and post-transcriptional mechanisms, such as microRNAs (miRNAs), are important in the regulation of pluripotency (Young, 2011). However, the mechanisms underlying the unique property of pluripotency are still not completely understood. In eukaryotic cells, three DNA-directed RNA polymerases, polymerases I, II, and III (Pol I–III), regulate transcription of different sets of target genes. Pol III regulates transcription of structural RNAs (tRNAs and 5S rRNAs) and several small non-protein-coding RNAs (ncRNAs) including miRNAs (Ozsolak et al., 2008; Borchert et al., 2006; Oler et al., 2010; Dieci et al., 2007). Pol III, like Pol I and Pol II, is composed of multiple subunits of which POLR3G (DNA-directed RNA polymerase III subunit, RPC32, RPC7) is a Pol III-specific subunit with no counterpart in Pol I or Pol II. POLR3G subunit is needed for transcriptional initiation of Pol III, thus being important for the proper function of this polymerase (Wang and Roeder, 1997).