Jung Jin Gyoung, Lee Young Mok, Park Tae Sub, Park Sang Hyun, Lim Jeong Mook, Han Jae Yong
Department of Food and Animal Biotechnology, Seoul National University, Seoul 151-921, Korea.
Biol Reprod. 2007 Jan;76(1):173-82. doi: 10.1095/biolreprod.106.056275. Epub 2006 Oct 11.
We recently succeeded in inducing germline transmission by transferring chicken testicular cells into heterologous testes. This study was designed subsequently to identify pluripotent cells in the testicular cells, which would induce the germline transmission. Testicular cells retrieved from juvenile (4-wk-old) or adult (24-wk-old) White Leghorn (WL) chickens were stained with germ cell-specific markers anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, and anti-ITGB1 antibodies; 2C9; and lectin-Solanum tuberosum agglutinin (STA). The percentages of the cells that were positive for each marker were within the ranges of 0.33% -0.44% and 0.029%-0.072% of the total testicular cell population in the juvenile and adult, respectively, and significant (P < 0.0002) differences were detected between the ages. When 1 x 10(6) testicular cells were cultured in Dulbecco minimum essential medium-based medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF2), and/or insulinlike growth factor 1 (IGF1), colony formation was detected only in LIF++FGF2-containing or LIF+FGF2+IGF1-containing medium during primary culture, and the supplementation of LIF+FGF2+IGF1 was the most efficient for maintaining the colony-forming cells through subculture. The established cells retrieved at the end of the primary culture or the 20th subpassage were positive for chicken germ cell-specific periodic acid-Schiff (PAS), EMA1, 2C9, SSEA1, SSEA3, SSEA4, ITGA6, and ITGB1; and lectin-STA markers (evaluated after 11th subpassage). Double staining of lectin-STA with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, and anti-ITGB1 also was possible. They differentiated spontaneously into embryoid bodies after being cultured in LIF-free medium. We conclude that germline stem cell-like cells are present in chicken testicular cells retrieved from both juvenile and adult testes, which can be identified with the specific markers for primordial germ cells or embryonic germ cells.
我们最近通过将鸡睾丸细胞移植到异种睾丸中成功诱导了种系传递。随后设计了这项研究，以鉴定睾丸细胞中的多能细胞，这些细胞可诱导种系传递。从幼年（4周龄）或成年（24周龄）白来航（WL）鸡中获取的睾丸细胞，用生殖细胞特异性标志物抗SSEA1、抗SSEA3、抗SSEA4、抗EMA1、抗ITGA6和抗ITGB1抗体；2C9；以及凝集素-马铃薯凝集素（STA）进行染色。每种标志物阳性的细胞百分比在幼年和成年睾丸细胞总数的0.33%-0.44%和0.029%-0.072%范围内，并且在年龄之间检测到显著（P<0.0002）差异。当1×10⁶个睾丸细胞在补充有白血病抑制因子（LIF）、碱性成纤维细胞生长因子（FGF2）和/或胰岛素样生长因子1（IGF1）的基于杜尔贝科最低必需培养基的培养基中培养时，仅在原代培养期间在含有LIF++FGF2或LIF+FGF2+IGF1的培养基中检测到集落形成，并且补充LIF+FGF2+IGF1对于通过传代培养维持集落形成细胞最有效。在原代培养结束时或第20次传代时获取的已建立细胞对鸡生殖细胞特异性过碘酸希夫（PAS）、EMA1、2C9、SSEA1、SSEA3、SSEA4、ITGA6和ITGB1呈阳性；以及凝集素-STA标志物（在第11次传代后评估）。凝集素-STA与抗SSEA1、抗SSEA3、抗SSEA4、抗ITGA6和抗ITGB1的双重染色也是可行的。它们在无LIF培养基中培养后自发分化为胚状体。我们得出结论，从幼年和成年睾丸中获取的鸡睾丸细胞中存在种系干细胞样细胞，这些细胞可用原始生殖细胞或胚胎生殖细胞的特异性标志物进行鉴定。
