A Comparative Analysis of Culture Systems with Human Amniotic  Mesenchymal Stem Cells

Mahdi  Jahanbakhsh; Fatemeh  Asgari; Roya  Hassani; Morteza  Koruji; Hamid Reza  Asgari

doi:10.18502/jri.v26i4.21086

Mahdi Jahanbakhsh Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
Fatemeh Asgari Avicenna Fertility Center, Avicenna Research Institute, ACECR, Tehran, Iran
Roya Hassani Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
Morteza Koruji Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
Hamid Reza Asgari Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/jri.v26i4.21086

Keywords: Amniotic mesenchymal stem cells, Differentiation, Placenta, Proliferation, Spermatogenesis.

Abstract

Background: A "indirect co-culture using mesh" 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 op timized environment.

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 con ditioned media, and SSCs indirectly co-cultured with hAMSCs. Gene expression an alyzed for Plzf, c-kit, Sycp3, and Prm1. Immunohistochemistry assessed Plzf, and flow cytometry evaluated c-kit and Plzf.

Results: Showed a twofold increase in Plzf-positive cells after 14 days of culture (76.47%, p≤0.05), with a significant elevation in Plzf gene expression observed in the conditioned media group (188.1±65%, p≤0.05). Conversely, the expression of the c-kit gene decreased significantly in both the conditioned media and "indirect co culture using mesh" 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.

Conclusion: Our results demonstrate that the inclusion of growth factors, such as GDNF and BMP-4, along with conditioned media and an "indirect co-culture using mesh" 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 "indirect co-culture using mesh" systems for promoting both the proliferation and differentiation of SSCs.