From single-cell to many cells

Researchers use RNA sequencing to investigate spermatogonial stem cells and tackle infertility

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SAN DIEGO—Spermatogenesis, or the production of sperm, is an extremely prolific process—in normal males, more than 1,000 sperm are generated per second. The cells driving this process are known as spermatogonial stem cells. However, in many men, the production of sperm does not occur at normal levels; more than 100 million men suffer from infertility globally, either as a result of genetics or as a side effect from chemotherapy. As such, spermatogonial stem cells are seen as as a potential target for remedying male infertility.
Unfortunately, efforts to grow spermatogonial stem cells in a laboratory setting have generally failed. According to Dr. Miles Wilkinson, professor in the Department of Obstetrics, Gynecology and Reproductive Sciences at the University of California, San Diego (UC San Diego) School of Medicine, this is due to a variety of factors.
“There's really two issues,” he tells DDNews. “One is to keep the spermatogonial stem cells alive. Our experience is that the cells with the characteristics of spermatogonial stem cells die after two or three weeks or so. This is something we are trying to improve on by changing culture conditions. The second problem is getting spermatogonial stem cells to expand in numbers. This is what you need to do ultimately for therapy: to go from a few cells—because spermatogonial stem cells seem to be relatively infrequent cells in the testes—to the point of having enough for therapeutic purposes. We and others in the field are working on this problem too.”
In addition, identification of spermatogonial stem cells is an issue, Wilkinson adds, noting that researchers are currently dependent on gene markers to determine which cell clusters consist of spermatogonial stem cells.
Wilkinson and his UC San Diego colleagues recently published work in Cell Reports, in a paper titled “The Neonatal and Adult Human Testis Defined at the Single-Cell Level,” seeking to narrow in on more specifics about spermatogonial stem cells. Their work focused on single-cell sequencing to explore the genetics of the stem cells.
“Single-cell RNA sequencing determines the activity of hundreds of genes in the genomes of single cells,” said Wilkinson. “Because each cell type has a different combination of active genes, this technique allows new cell types to be identified. Applying this approach to the testis, we uncovered many different stages of sperm precursor cells in human testes.”
The team identified cell subtypes that potentially include spermatogonial stem cells, as well as biomarkers that define the stem cells. By analyzing neonatal testes, they also pinpointed “cell populations with characteristics of primordial germ cells (PGCs)” and were able to “map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult [spermatogonia] stages,” the authors noted.
The other key issue in the field, Wilkinson notes, is to figure out which cells with the characteristics of spermatogonial stem cells (SSCs) are actually stem cells. He tells DDNews that they intend to use a “functional stem cell test” to determine which of the various clusters of germ cells that they identified by single-cell RNA sequencing (as described in the recent paper) actually harbor the stem cells.
“Our paper is helping us, because the genes that are expressed in the cells next to the germ cells—we call those somatic cells—are making factors, proteins, that are critical for the germ cells to survive and proliferate,” says Wilkinson. “We identified a lot of these genes that we think are important for that. So now what we're doing is trying to grow the human SSCs with those proteins. Our single-cell sequencing analysis was critical because it gave us lots of candidate proteins we can try, looking for the holy grail, the key one or two proteins that would allow for survival and expansion of SSCs.”

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