In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix

Abstract Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three‐dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1‐positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two‐dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35‐day mark, the percentage of Prm1‐positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio‐scaffold for enhancing mouse in vitro spermatogenesis.