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: The co-culture of spermatogonial stem cells (SSCs) with human amniotic mesenchymal stem/stromal cells (hAMSCs) may support in vitro spermatogenesis. This study aimed to evaluate SSC proliferation and differentiation in optimized culture systems.

Methods: SSCs from 3–6-day-old mice (n=10) were indirectly co-cultured with human placental tissue–derived hAMSCs via a mesh system and evaluated after a two-week culture period. The evaluation included three experimental groups: a control group, SSCs cultured with conditioned media, and SSCs indirectly co-cultured with hAMSCs via a mesh. Gene expression analysis was conducted for Plzf, c-kit, Sycp3, and Prm1, with immunohistochemistry performed to assess Plzf marker expression. Additionally, flow cytometry was utilized to evaluate c-kit and Plzf markers.

Results: Plzf-positive cells doubled after 14 days (76.47%, p≤0.05), with Plzf gene expression significantly increased in the conditioned media group (188.1±65%, p≤0.05). c-kit expression decreased in both conditioned media and mesh-based co-culture groups. Sycp3 and Prm1 expression also increased in the conditioned media group compared to 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 BMP4, together with conditioned media and an indirect mesh-based co-culture system, significantly enhances SSC proliferation and differentiation. The op-timized conditioned media provided by hAMSCs serve as a superior feeder com-pared to traditional indirect mesh-based co-culture systems for promoting both SSC proliferation and differentiation.