MicroRNA Analysis of In Vitro Differentiation of Spermatogonial Stem Cells Using a 3D Human Testis Organoid System

Area of Science:

• Reproductive Biology
• Molecular Biology
• Stem Cell Science

Background:

• Spermatogenesis, the production of male gametes from spermatogonial stem cells (SSC), is crucial for reproduction but its biochemical processes are not fully understood.
• MicroRNAs (miRNAs) are key post-translational regulators of gene activity, yet their specific role in spermatogenesis remains largely uncharacterized.
• Advancements in laboratory techniques enable long-term SSC culture and in vitro spermatogenesis, necessitating a deeper understanding of the underlying molecular mechanisms.

Purpose of the Study:

• To characterize the presence and expression profiles of miRNAs in a 2D SSC culture system and a 3D human testis organoid (HTO) model.
• To compare miRNA expression between native whole testis (WT) tissue and progressively complex in vitro testicular cell culture stages.
• To identify conserved and unique miRNAs across different conditions to elucidate their potential roles in spermatogenesis.

Main Methods:

• Testicular cells were isolated from three brain-dead subjects and cultured for 4-5 weeks to form 3D HTOs.
• RNA was extracted from whole testis (WT), 2D SSC culture (HTO Day Zero), Day 2 HTOs, and differentiated Day 23 HTOs.
• miRNA expression was analyzed using the Nanostring nCounter miRNA panel to identify expression patterns and differences.

Main Results:

• A total of 195 miRNAs were expressed in WT, 186 in 2D culture, 190 in Day 2 HTOs, and 187 in differentiated HTOs.
• 133 miRNAs were common across all conditions, while unique miRNAs were identified for each condition (41 in WT, 17 in 2D, 6 in Day 2 HTOs, 11 in differentiated HTOs).
• Twenty-two miRNAs showed similarity between WT and differentiated HTOs, indicating some conservation of expression.

Conclusions:

• Significant variations in miRNA expression exist between native testicular tissue and in vitro culture systems, including 3D HTOs.
• Despite variations, certain miRNAs are preserved across different conditions, suggesting their potential importance in maintaining aspects of spermatogenesis.
• These findings provide valuable data for understanding spermatogenesis in vitro and may guide improvements in laboratory-based reproductive technologies and research.