Identification, culture, and characterization of germline stem cell-like cells in chicken testes.
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.
Jung
Jin Gyoung
JG
Department of Food and Animal Biotechnology, Seoul National University, Seoul 151-921, Korea.
Lee
Young Mok
YM
Park
Tae Sub
TS
Park
Sang Hyun
SH
Lim
Jeong Mook
JM
Han
Jae Yong
JY
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D002645:Chickens; D005854:Germ Cells / Q000737:chemistry / Q000166:cytology; D008297:Male; D039904:Pluripotent Stem Cells / Q000737:chemistry / Q000166:cytology*; D013094:Spermatozoa / Q000737:chemistry / Q000166:cytology*; D013737:Testis / Q000737:chemistry / Q000166:cytology*
D000954:Antigens, Surface; D015415:Biomarkers