Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro

Abstract

Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis.

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

“…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).…”

“…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). Update, the effects of cytokines and growth factors on porcine mGSCs were not clear (Goel et al 2009;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). 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.…”

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro

“…The expression of these genes may be required for the survival and proliferation of gonocytes. In the pig, upregulation of pluripotency-related genes in gonocytes in primary culture was shown to stimulate the proliferation and stem cell potential of the gonocytes (Goel et al 2009), suggesting that DBA-coated plates can support the proliferation and survival of cultured bovine germ cells. Meanwhile, the expression of NANOG on the DBA-precoated plates was decreased, whereas UCHL1 expression increased.…”

“…To achieve this, the expression of vital pluripotency-related genes, such as NANOG and POU5F1, is essential, but their expression gradually decreases as the passage number increases (Goel et al 2009). The pluripotent state in cultured germ cells can be supported by using ECM components that interact with adhesion molecules on the cell surface (Chai and Leong 2007), which suggests that some cell surface molecules can regulate the expression of genes associated with a pluripotent state in cultured germ cells.…”