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Volume 23, Issue 3 (May 2021) 23, 240–248; 10.4103/aja.aja_95_20

Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis

Guo-Ping Mao2,3,4, Ming-Hui Niu2, Ying-Hong Cui1, Rui-Ling Tang1, Wei Chen1, Bang Liu1, Zuping He1,2

1 The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Changsha 410013, China
2 Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
3 The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China
4 Shanghai Key Laboratory of Embryo Original Diseases, Shanghai 200030, China

Correspondence: Dr. Z He (zupinghe@hunnu.edu.cn)

Date of Submission 25-Sep-2019 Date of Acceptance 02-Dec-2020 Date of Web Publication 29-Jan-2021


Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-KitW/W (W) mutant mice. Collectively, GFRA1-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo. This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.

Keywords: characterization; isolation and culture; Macaca fascicularis; spermatogonial stem cells; transplantation and transcriptomes

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