US20260176579
2026-06-25
Chemistry; metallurgy
C12N5/061
The patent application describes an innovative method for generating human spermatozoa in vitro from differentiating spermatogonia (dSPGs) and spermatogonial stem cells (SSCs). The process involves a sequential cell culture system with distinct, stage-specific culture media and conditions that allow the complete progression of human spermatogenesis outside the body. This method is groundbreaking as it enables the full in vitro recapitulation of human spermatogenesis, providing a valuable platform for research, infertility treatment, and assisted reproductive technologies.
The method involves three main stages: inducing entry of dSPGs and SSCs into meiosis, supporting meiotic progression to round spermatids, and promoting spermiogenesis to form elongated spermatozoa. Each stage utilizes specific culture media compositions and conditions to facilitate these transitions. The invention includes compositions, systems, and protocols for each stage, along with markers and methods for identifying successful progression through each developmental transition.
The first culture system involves culturing human dSPGs or SSCs in a nutrient-restricted medium with specific concentrations of retinoic acid, BMP proteins, activin A, insulin, and IWR-endo-1. This stage is conducted at a temperature of 31°C to 33°C for one to four days, facilitating the entry of cells into meiosis. Successful entry is indicated by EdU incorporation and specific markers such as HORMAD1 and γH2AX.
In the second stage, cells are cultured in a nutrient-rich medium with the addition of MEF feeder cells, FSH, testosterone, bovine pituitary extract, and optionally KITLG. The culture is maintained at 31°C to 33°C for eight to twelve days, allowing the progression of spermatocytes through meiosis to form round spermatids. The appearance of cells expressing ACRV1 indicates successful progression.
The final stage involves culturing round spermatids in a nutrient-rich medium with 2-mercaptoethanol, testosterone, retinoic acid, and MEF-conditioned media. This stage is conducted at 31°C to 33°C for five to eight days, resulting in the generation of flagellar cells, which are indicative of mature spermatozoa. This comprehensive system provides a robust framework for studying and applying human spermatogenesis in vitro.