en <p>Background: A &ldquo;indirect co-culture using mesh&rdquo; system is commonly employed to maintain spermatogenesis in cancer patients undergoing chemotherapy and radiation. This study aimed to investigate the co-culturing of mouse spermatogonial stem cells (SSCs) with human amniotic mesenchymal stem/stromal cells (hAMSCs) in an optimized environment.<br /> Methods: SSCs from 3-6-day-old mice (n=10) were indirectly co-cultured with hAMSCs via mesh for two weeks. Three groups evaluated: control, SSCs with conditioned media, and SSCs indirectly co-cultured with hAMSCs. Gene expression analyzed for Plzf, c-kit, Sycp3, and Prm1. Immunohistochemistry assessed Plzf, and flow cytometry evaluated c-kit and Plzf.<br /> Results: Showed a twofold increase in Plzf-positive cells after 14 days of culture (76.47%, P&le;0.05), with a significant elevation in Plzf gene expression observed in the conditioned media group (188.1 &plusmn; 65%, P&le;0.05). Conversely, the expression of the c-kit gene decreased significantly in both the conditioned media and &ldquo;indirect co-culture using mesh&rdquo; groups. Notably, Sycp3 and Prm1 expression levels significantly increased in the conditioned media group compared to the control. These findings suggest the potential of conditioned media as a novel feeder for promoting in vitro mouse spermatogenesis.<br /> Conclusion: Our results demonstrate that the inclusion of growth factors, such as GDNF and BMP-4, along with conditioned media and an &ldquo;indirect co-culture using mesh&rdquo; system utilizing meshes with SSCs, significantly enhances SSC proliferation and differentiation. The optimized conditions media provided by hAMSCs offer a superior feeder compared to traditional &ldquo;indirect co-culture using mesh&rdquo; systems for promoting both the proliferation and differentiation of SSCs.</p> Amniotic mesenchymal stem cells, Differentiation, Placenta, Proliferation 2013 2025 https://www.jri.ir/article/140285 https://www.jri.ir/documents/fullpaper/en/140285.pdf MahdiJahanbakhshStem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran122939FatemehAsgariAvicenna Fertility Center, Avicenna Research Institute, ACECR, Tehran, Iran122940RoyaHassaniDepartment of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran122941MortezaKorujiDepartment of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran1656Hamid RezaAsgariDepartment of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iranasgari.hr@iums.ac.ir61987