Long-term Culture And Identification Of The Spermatogonial Stem Cells Of Macaca Fascicularis

Objective To establish a method system of separation, purification, long-term culture in vitro, identification with marker, long-term frozen storage and physiological function analysis for spermatogonial stem cells (SSCs) of Macaca fascicularis, constructing a te chnical platform for application research of Macaca fascicularis SSCs.Methods Testes extracted from a two-year old Macaca fascicularis were processed with three-step enzyme digestion to produce suspension of cells, then SSCs were enriched by culturing the suspension of cells with differential adherence method. The enriched SSCs were then co-cultured over20days with mitomycin-treated STO cells serving as trophoblast cells in serum free medium added with three important growth factors of GDNF, bFGF and GFRal. The generation of SSCs were tentatively identified with CDH1immunofluorescence staining. The acquired Macaca fascicularis SSCs were continuously cultured and passaged to the22th generation, then stored in liquid nitrogen for two years. The two-year stored cells were recovered and identified through immunofluorescence double staining GFRal with CDH1, Thy-1, Oct4or PLZF and RT-PCR amplifying markers above. Morphology and location of SSCs in Macaca fascicularis testis were manifested through immunofluorescence staining the cryosections of Macaca fascicularis testis tissue. The cloned cluster identified as SSCs were cultured in medium containing SCF factor to induce differentiation in vitro, and whether SSCs successfully differentiated into spermatocytes were evaluated with spermatocyte specific marker of Scp3by immunofluorescence staining.Results (1) the enriched cells began to form small clones after two-day culture, and these clones enlarged to remarkable size five days later. After20days culture, clusters of grape like cells appeared, which was consistent with morphology features of SSCs, and immunofluorescence staining demonstrated these cells express SSCs marker of CDH1.(2) Four groups of immunofluorescence double staining GFRal with CDH1, Thy-1, Oct4or PLZF demonstrated that all these SSCs specific markers expressed in the22th generation of Macaca fascicularis SSCs.(3) Results of RT-PCR also demonstrated that GFRal, CDH1, Thy-1, Oct4and PLZF expressed in these cells.(4) Immunofluorescence staining results of Macaca fascicularis testis showed that GFRα1expressed in single or paired cells adjacent to basement membrane.(5) microscopic observation showed that spermatocytes-like cells appeared on the eighth days later in cells derived from SSCs that were cultured in medium for inducing differentiation experiment, and immunofluorescence staining demonstrated that spermatocyte specific marker of Scp3expressed in these cells. Conclusions (1) A method system consisting of separation, purification, long-term in vitro culture, identification, long-term frozen storage and physiological function analysis for spermatogonial stem cells (SSCs) of Macaca fascicularis has been successfully established.(2) GFRal, CDH1, Thy-1and Oct4can be used for SSCs identification.(3) immunofluorescence staining cryosections of Macaca fascicularis testis tissue supports that GFRal is a reliable marker of Macaca fascicularis SSCs.(4) the long-term in vitro cultured SSCs can successfully differentiate into spermatocyte.