Abstract
BackgroundTo isolate and characterization of human spermatogonial stem cells from stem spermatogonium.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.
Highlights
To isolate and characterization of human spermatogonial stem cells from stem spermatogonium
Spermatogenesis begins with self-renewal and differentiation of spermatogonial stem cells (SSCs)
Our results indicate that two-step enzymatic digestion is a good method to isolate testis cells
Summary
To isolate and characterization of human spermatogonial stem cells from stem spermatogonium. The incidence of male infertility such as aspermia, oligospermia and asthenospermia of unknown origin has been increasing in recent years. Investigations on the mechanism of spermatogenesis and its influencing factors have become one of the hot topics in andrology. Spermatogenesis begins with self-renewal and differentiation of spermatogonial stem cells (SSCs). The complexity of the systemic and testicular microenvironment makes it difficult to conduct in vivo study on of SSCs. the establishment of stable human SSCs in vitro would provide a useful stem cell model for studying the proliferation and differentiation of SSCs. The
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