Male infertility is where a man has a lower chance of impregnating his female partner if at all, leading to exhausting, and sometimes disappointing, treatment involving artificial or in vitro fertilization techniques. Infertility affects one in seven of all men globally, with abnormal semen listed as the main cause of this condition. A multitude of issues known to affect semen quality exists presently, with much research centered around improving the production and activity of sperm.
Towards growing sperm in the lab
Sperm are generated from male spermatogonial stem cells (SSCs) differentiated from spermatogonia precursor cells in the testes in a process known as spermatogenesis, estimated to produce more than 1,000 sperm per second in normal males.
One idea for treating male sterility is SSC therapy, where these stem cells are differentiated into healthy sperm in the laboratory. These cells, however, are incredibly difficult to distinguish in testicular tissue leading to incorrectly identified cells being cultured.
Now, a study from researchers at the University of California San Diego develops a reliable method for identifying and culturing cells matching the characteristics of human SSCs. The team states their approach, based on single-cell RNA-sequencing analysis, is a significant step toward bringing SSC therapy into the clinic. The study is published in the Proceedings of the National Academy of Sciences.
Previous studies have indicated spermatogenesis depends on a series of events in germ cells, known as spermatogonia, which differentiate into self-renewing SSCs. Recent studies from the group analyzed adult testes revealing four undifferentiated spermatogonia colonies, each of which expressed specific marker genes.
The lab then went on to identify protein markers for the most primitive spermatogonia state, allowing the purification of this potentially SSC-enriched cell colony.
Their study also identified definitive biomarkers for SSCs, detected using specific antibodies, to enable the accurate identification and capture of these stem cells. The current study developed an approach to purify human primitive undifferentiated spermatogonia whose signaling pathways were altered to produce and culture SSCs for therapeutic purposes.
A viable therapy for infertile men
The current study utilizes single-cell RNA sequencing and germ-cell transplantation to identify and purify human SSCs from 30 testes biopsies. The team then gathered the profile of genes expressed in these human SSCs to reveal the best conditions to support their growth in the lab.
Results identify a cell-surface protein, called PLPPR3, enabling purification of human primitive undifferentiated spermatogonia highly enriched for SSCs. Data findings show RNA-sequencing analysis of these enriched SSCs with differentiating spermatogonia identifies genes encoding the TGF-β, GDNF, AKT, and JAK-STAT developmental signaling pathways.
The lab then manipulated these signaling pathways to demonstrate that GDNF broadly supports human spermatogonia culture, while activin-A selectively supports more advanced human spermatogonia. Results show AKT pathway inhibition supports the culture of primitive human undifferentiated spermatogonia.
The group explains this strongly suggested the inhibition of the AKT pathway, a cellular system controlling cell division and survival, could be used to culture human SSCs in vitro for therapeutic applications.
The lab confirms AKT inhibitors maintains human SSCs by inhibiting the development of later-stage sperm precursors, capable of supporting the culture of human cells with the molecular characteristics of SSCs for two-to-four weeks.
The team surmises they successfully cultured human spermatogonial stem cells for therapeutic applications. For the future, the researchers state they will investigate how to maintain SSCs longer in culture so they might be clinically viable.
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