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Updated: May 11, 2026
Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines
Published on: February 25, 2013
Isolation and culture of human spermatogonial stem cells derived from testis biopsy
Leila Goharbakhsh1, Arash Mohazzab, Sheida Salehkhou
1Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran ; Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Background:
In cancer patients, chemo and radiotherapy can cause infertility by damaging spermatogenesis process. This process is based on self-renewal and differentiation of a rare population of the testicular cells called Spermatogonial Stem Cells (SSCs). Scientists have tried to isolate, enrich and culture Human spermatogonial stem cells, hoping to resolve infertility problems in cancer recovered patients in the future.
Methods:
Spermatogonial stem cells were isolated and purified from human testicular biopsies sample consisting of at least 500,000 and at most 2,000,000 cells. Two enzymatic digestion steps were performed. Enriching methods, differential plating, and specific culture in serum-free medium with added growth factors: human GDNF, bFGF, EGF and LIF was performed on coated dishes.
Results:
Human spermatogonial stem cell clusters were observed after 7 to 10 days in specific culture, then after several passages and successful expanding duration of 52 days, the cells were evaluated by three layer immunocytochemistry test (LSAB) to stain GPR125 protein as a surface marker in human spermatogonial stem cells.
Conclusion:
In current study human spermatogonial stem cell were isolated and expanded with the least manipulations in comparison with the other usual isolation methods like florescent or magnetic activated cell sorting. In contrast to the other SSCs isolation and culture methods, this system is based on the testicular biopsies against large samples, thus suggested method in this study is closer to clinical usage in the future.
Insights
Researchers developed a simplified method to isolate and expand human spermatogonial stem cells (SSCs), offering hope for future fertility restoration in cancer survivors. This technique minimizes manipulation for potential clinical application.
Area of Science:
- Reproductive biology
- Stem cell research
- Oncofertility
Background:
- Chemotherapy and radiotherapy can cause infertility in cancer patients by damaging spermatogenesis.
- Spermatogonial stem cells (SSCs) are crucial for sperm production through self-renewal and differentiation.
- Current research aims to isolate, enrich, and culture human SSCs for future fertility restoration.
Purpose of the Study:
- To develop a simplified method for isolating and expanding human spermatogonial stem cells (SSCs).
- To establish a culture system for SSCs with minimal manipulation for potential clinical use.
Main Methods:
- Human testicular biopsies were processed using enzymatic digestion.
- Cells were enriched via differential plating and cultured in serum-free medium with specific growth factors (GDNF, bFGF, EGF, LIF).
- Culture dishes were coated, and cells were maintained for up to 52 days.
Main Results:
- Human spermatogonial stem cell clusters formed within 7-10 days of culture.
- Cells were successfully expanded for 52 days through multiple passages.
- Immunocytochemistry confirmed GPR125 as a surface marker on the cultured SSCs.
Conclusions:
- The study presents a method for isolating and expanding human SSCs with fewer manipulations than traditional methods like cell sorting.
- This approach utilizes testicular biopsies, making it more amenable to clinical application compared to methods requiring larger samples.
- The developed system offers a promising step towards clinical fertility preservation strategies for cancer survivors.
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