Isolation and characterization of human spermatogonial stem cells.
BACKGROUND
To isolate and characterization of human spermatogonial stem cells from stem spermatogonium.


METHODS
The disassociation of spermatogonial stem cells (SSCs) were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4)-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established.


RESULTS
The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells.


CONCLUSIONS
The two-step enzyme digestion (by type I collagenase and trypsin) process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.
Department of Urology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Shixue
S
Liu
Ziwei
Z
Tang
Tao
T
Xiong
Wei
W
Tang
D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D017364:Collagenases / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D015203:Reproducibility of Results; D012708:Sertoli Cells / Q000166:cytology; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D015415:Biomarkers; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D014357:Trypsin; D017364:Collagenases; C417249:collagenase 1