title: Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes.
abstract: OBJECTIVE
  To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes.
  
  
  METHODS
  We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method.
  
  
  RESULTS
  Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn't and was detected, respectively.
  
  
  CONCLUSIONS
  We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.
authors.affiliation: Department of Animal Life Science, Kangwon National University, Chuncheon, 200-701, South Korea.
authors.forename: Min Hee
authors.initials: MH
authors.lastname: Park
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Park
authors.forename: Min Seong
authors.initials: MS
authors.lastname: Kim
authors.forename: Kwon Young
authors.initials: KY
authors.lastname: Lee
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Park
authors.forename: Jung Im
authors.initials: JI
authors.lastname: Yun
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Choi
authors.forename: Eun song
authors.initials: Es
authors.lastname: Lee
authors.forename: Seung Tae
authors.initials: ST
authors.lastname: Lee
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry; D008297:Male; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013552:Swine; D013737:Testis / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers