An Alternative Long‐Term Culture System for Highly‐Pure Mouse Spermatogonial Stem Cells

Abstract

Increasing evidence suggests that spermatogonial stem cells (SSCs) have great clinical potential to give rise to a variety of cell types besides all spermatogenic lineage cells. The development of an efficient method for long-term culture of highly-pure SSCs is essential for further studies related to SSC biological events. Here, we describe an in vitro culture system obtaining mouse SSC cultures of high purity, viability, and proliferation. For establishing long-term cultures of SSCs, we mainly focused on isolation procedures and culture conditions. These included co-coating of extracellular substrates, that is, poly-L-lysine (PLL) and laminin, as well as combinatiorial use of three milder enzymes and simultaneously less trypsin to minimize enzyme-mediated degradation of SSCs. Furthermore, a unique purification procedure was performed to effectively eliminate contaminating non-SSCs. Finally, a critical step is to ensure SSC maintenance and expansion by utilizing optimal culture medium. Obtained data suggest that applying our optimally modified method, SSCs can be cultured for over 90 days with high purity (around 93.5%). Moreover, SSCs isolated and expanded using our protocol fulfills all criteria of SSCs without losing their stemness-characterized by SSC-phenotypic gene expression and long-term self-renewal. This study describes for the first time a protocol allowing isolation and expansion of SSCs suitable for numerous studies related to SSC-based clinical therapies of various diseases.

p53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly‐L‐lysine

“…GDNF has a critical role in mGSCs self‐renewal, mediated by GFRa1 and C‐Ret (Meng et al, ; Naughton, Jain, Strickland, Gupta, & Milbrandt, ; Oatley et al, ), through the activation of SFK and AKT pathways (He, Jiang, Hofmann, & Dym, ; Lee et al, ; Oatley et al, ). However, whether PL regulates mGSCs self‐renewal and differentiation through any of the aforementioned pathways is yet to be determined (He et al, ; Sahare et al, ). In the dairy goat, studies on mGSCs were mainly based on cell culture and focused on description of biological characteristics (Hua et al, ).…”

“…Poly‐L‐lysine, a synthetic molecule with a highly positively charged homopolypeptide, enhances cell adhesion by imparting a positive charge to the culture surface and is often used as a coating agent in different applications (Lam & Longaker, ). In addition, PL was reported to benefit the differentiation of neural stem cells, mesenchymal stem cells, pluripotent stem cells (PSCs), spermatogonia stem cells (SSCs), as well as in tissue engineering (He, Lu, Zhang, Hao, & Yang, ; Lam & Longaker, ; Lee, Wu, Cheng, & Yang, ; Malik et al, ; Sahare et al, ; Zhang et al, ). However, the commonly used extracellular matrices in cell culture, including gelatin, laminin, Matrigel, fibronectin and poly‐lysine, have rarely been compared for their effects on mGSC cultures.…”

Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro

“…Secondly, SSCs are difficult to identify and purify due to a lack of SSC-specific markers and possibly due to the existence of subpopulations of SSCs in various species [Albert et al 2012;Izadyar et al 2011;Kossack et al 2013]. Thirdly, current strategies focusing on exvivo SSC expansion are complex, time-and resourceconsuming and often result in low cell purity and vitality [He et al 2015].…”

“…However, these sophisticated manipulations rely on markers that are not specific for SSCs or cross-react with other epitopes and often result in substantial cell death and a lack of available SSCs for further culture [Valli et al 2014a]. Furthermore, it should be noted that various culture conditions have been shown to promote SSC expansion including feeder vs. non-feeder cultures and use of several different media amongst others [He et al 2015].…”

“…Our knowledge of SSC biology arises mostly from studies in rodents [Boitani et al 2016]. While long term SSC proliferation among human samples is debatable, murine SSC expansion over months in vitro has been reported by multiple researchers [Kanatsu-Shinohara et al 2005;Kanatsu-Shinohara et al 2003;Seandel et al 2007] and murine models have been used extensively for SSC culture studies focusing on novel approaches for in vitro SSC propagation [He et al 2015;Kanatsu-Shinohara et al 2003] and differentiation [Abu Elhija et al 2012]. It should be noted that most murine SSC culture experiments included juvenile mice whose testes have a relatively high SSC concentration prior to initiation of spermatogenesis [Abu Elhija et al 2012;He et al 2015].…”