Cryopreserved ovine spermatogonial stem cells maintain stemness and colony forming ability in vitro

Abstract

Objective: To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells (SSCs) in vitro.

Methods: Sheep testicular cells were isolated and putative SSCs were enriched by the laminin-based differential plating method. Putative SSCs were co-cultured with the Sertoli cell feeder prepared by the Datura Stramonium Agglutinin (DSA-lectin)-based method. The cultured putative SSCs were cryopreserved in Dulbecco's Modified Eagle Medium-10% fetal bovine serum mixture (DMEM-10% FBS) media containing 10% dimethyl sulfoxide (DMSO) alone or 10% DMSO plus 200 mM trehalose. Cryopreserved putative SSCs were evaluated for their proliferation potential using in vitro culture and stemness by immunocytochemistry. Finally, the transfection ability of cryopreserved putative SSCs was analyzed.

Results: We isolated 91% viable testicular cells from sheep testes. The majority of the laminin enriched cells expressed the SSC related marker, ITGA6. Co-culture of sheep putative SSCs with Sertoli cell feeder resulted in the generation of stable colonies, and the expression of SSC marker was maintained after several passages. A significantly higher number of viable putative SSCs was recovered from SSCs cryopreserved in media containing 10% DMSO and 200 mM trehalose compared to 10% DMSO alone (P<0.01). Cryopreserved putative SSCs formed colonies and showed SSC marker expression similar to the non-cryopreserved putative SSCs. The appearance of green fluorescent colonies over the Sertoli cell feeder indicated that cryopreserved sheep SSCs were successfully transfected.

Conclusions: Cryopreserved putative SSCs can retain their stemness, colony forming ability, and transfection efficiency in vitro. Our research may help in the effective preservation of germplasm and the generation of transgenic ovine species.

Keywords

Spermatogonial stem cell / Sheep / Stemness / Cryopreservation / Transfection

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors thank the Directors of ICMR-National Animal Resource for Biomedical Research, Hyderabad, Govt. of India; and Inter University Center for Biomedical Research, Kerala, India.

Funding

This study received no extramural funding.

Authors’ contributions

R. Kumar Pramod designed the study, conducted experiment, wrote the manuscript and helped in analysis and interpretation of the data. Deepthi Varughese helped in experiment and conducted the data analysis. A. Javed Jameel helped in both interpretation of data and manuscript editing. Bhisma Narayan Panda helped in experiment and manuscript writing. Soma Goswami helped in manuscript writing. Abhijit Mitra was involved in critical reading of the manuscript. All authors read and approved the final manuscript.

Publisher's Note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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