Abstract: A sequential cell culture system and methods for the in vitro generation of human spermatozoa from differentiating spermatogonia (dSPGs) and spermatogonial stem cells (SSCs) is provided. The instant disclosure encompasses the discovery and optimization of distinct, stage-specific culture media and conditions that enable, for the first time, the complete progression of human spermatogenesis outside the body. The process includes: (1) inducing entry of dSPGs and SSCs into meiosis; (2) supporting meiotic progression and completion to round spermatids; and (3) promoting spermiogenesis and the formation of elongated, spermatozoa. The invention further provides compositions, systems, and protocols for each stage, as well as markers and methods for identifying successful progression through each developmental transition.

Abstract: The present disclosure provides an iterative process for identifying culture conditions that maintain identity, growth, and survival of testicular cells in vitro. Testicular cell culturing systems for supporting human spermatogenesis and culture using identified culture conditions are also provided. The methods and the culture conditions can be used to culture healthy and viable spermatozoa with lower rates of deleterious or de novo mutations or epigenetic perturbations from fertile and infertile men for future use with assisted reproductive technologies.