In vitro propagation of male germline stem cells from piglets

Abstract

Purpose To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. Methods Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. Results The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10-ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population.Conclusions In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

“…The authors evaluated the effects of growth factors based on the number of colonies of SSC-like, and expression level of SSC markers and suggest that FGF can impede successful derivation of porcine SSCs from neonate pig testis (Goel et al 2009). Update, the effects of cytokines and growth factors on porcine mGSCs were not clear (Goel et al 2009;Zheng et al 2013). In the present study, the pmGSCs have been cultured up to 14 passages for over two months, while still maintained a typical morphology to mouse and human GSCs, indicating that our cultural system could simulate the cellular niche in seminiferous tubules that maintained the proliferation and differentiation of mGSCs in vitro.…”

“…Previous studies demonstrated that several pluripotent/multipotent germline stem cells have been established and characterized by different research groups using various protocols, and murine and human male GSCs can be cultured for long periods and retain the capacity to differentiate into multiple cell types in the presence of serum or feeder cells in vitro. Recently, porcine, goat and bovine mGSCs were cultured and characterized, and these cells shared similar characteristics of ES cells and GSCs (Kanatsu-Shinohara et al 2008;Goel et al 2009;Kuijk et al 2009;Hua et al 2011;Zeng et al 2013;Zheng et al 2013;Park et al 2014). However, the methods available for isolating SSCs and mGSCs from porcine testicular cells have a low efficiency of cell separating, and it is difficult to establish a stable livestock mGSCs line Zheng et al 2013;Park et al 2014).…”

“…Recently, porcine, goat and bovine mGSCs were cultured and characterized, and these cells shared similar characteristics of ES cells and GSCs (Kanatsu-Shinohara et al 2008;Goel et al 2009;Kuijk et al 2009;Hua et al 2011;Zeng et al 2013;Zheng et al 2013;Park et al 2014). However, the methods available for isolating SSCs and mGSCs from porcine testicular cells have a low efficiency of cell separating, and it is difficult to establish a stable livestock mGSCs line Zheng et al 2013;Park et al 2014). In this study, we used Laminin in combination with differentiating plating (DP) method to isolate SSCs from postnatal porcine testicular cells.…”

“…GDNF is secreted by the somatic Sertoli cells and peritubular cells, and is a well-known inducer of mouse SSC proliferation in vitro (Oatley et al 2008). Binding of GDNF to the GDNF-family receptor α1 (GFRα1) catalyses the activation of the c-RET receptor, which activates several signal cascades, such as PI3K/AKT, MEK and SCR kinases (Zheng et al 2013). GDNF activation upregulates expression of the transcription factor-encoding genes Bcl6b, Etv5, and Lhx1, which influence mouse SSC selfrenewal (Oatley and Brinster 2008).…”

“…EGF and FGF had a positive effect on the number and size of the SSC-like colonies in primary cultures of neonate pig testis. In vitro, propagation of porcine mGSCs could be maintained in the presence of 1% FBS and supplementation of growth factors for one month (Zheng et al 2013). The authors evaluated the effects of growth factors based on the number of colonies of SSC-like, and expression level of SSC markers and suggest that FGF can impede successful derivation of porcine SSCs from neonate pig testis (Goel et al 2009).…”