Isolation and characterization of human spermatogonial stem cells

Autor: Zi-wei Tang, Tao Xiong, Wei Tang, Shi-xue Liu
Jazyk: angličtina
Předmět:
Male
Stage-Specific Embryonic Antigens
endocrine system
Octamer Transcription Factor-3
lcsh:QH471-489
Cell Survival
Cell Culture Techniques
andrology
Biology
lcsh:Gynecology and obstetrics
male infertility
Flow cytometry
Endocrinology
spermatocytes
Testis
medicine
Humans
lcsh:Reproduction
Trypsin
Collagenases
Fluorescent Antibody Technique
Indirect
Cells
Cultured
lcsh:RG1-991
Cell Proliferation
spermatogonial stem cells
Spermatogonium
Sertoli Cells
medicine.diagnostic_test
Research
Stem Cells
Reproducibility of Results
Obstetrics and Gynecology
Flow Cytometry
Sertoli cell
Immunohistochemistry
Molecular biology
Coculture Techniques
spermatogenesis
spermatogonia
medicine.anatomical_structure
Reproductive Medicine
Cell culture
sperm cells
Stem cell
Spermatogenesis
Biomarkers
Developmental Biology
Zdroj: Reproductive Biology and Endocrinology, Vol 9, Iss 1, p 141 (2011)
Reproductive Biology and Endocrinology : RB&E
ISSN: 1477-7827
DOI: 10.1186/1477-7827-9-141
Popis: 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.
Databáze: OpenAIRE
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