Three-dimensional Co-culture of Human Spermatogonial Stem Cells with Sertoli Cells in a Soft Agar Culture System Supplemented by Growth Factors and Laminin

Background: Three-dimensional (3-D) culture system for in vitro proliferation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility, particularly in prepubertal cancer patients. The purpose of this study...

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Bibliographic Details
Published in:Iranian journal of medical sciences 2023-01, Vol.48 (S1), p.129
Main Authors: Jabari, Ayob, Khadivi, Farnaz, Abbasi, Mehdi
Format: Article
Language:English
Subjects:
Integrins
Laminin
Medical research
Medicine, Experimental
Stem cells
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Summary:Background: Three-dimensional (3-D) culture system for in vitro proliferation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility, particularly in prepubertal cancer patients. The purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in a soft agar culture system (SACS) supplemented by Laminin and growth factors. Methods: Testicular cells were isolated from the testes of brain-dead patients and cultured in a two-dimensional (2-D) culture system for three weeks. After three weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. SSCs and Sertoli cells were transferred to the upper layer of SACS for three weeks. The number of colonies and expression of specific SSCs and Sertoli cell markers and apoptotic genes was evaluated. Results: Transplanted SSCs migrated into the basement membrane of seminiferous tubules of recipient mice. Expression of PLZF, [alpha]6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2-D and 3-D culture systems. The expression rate of PLZF, [alpha]6-Integrin, Bc[l.sub.2], and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P[less than or equal to]0.01). The expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P[less than or equal to]0.05). The three-D co-culture system decreased apoptosis and increased the propagation of human SSCs. Conclusion: This designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermia patients. Keywords * Coculture techniques * Adult germline stem cells * Laminin * Stem cells * Cell proliferation
ISSN:0253-0716