The first single-cell analysis of the human ovarian cortex revealed six main types of cells, but none of the oogonial stem cells that other researchers say they have isolated, according to a study published earlier this week (March 2) in Nature Communications. These findings are backed by the most advanced technologies, the authors say, and could put to rest a heated debate about the properties of the adult ovary that has raged for more than a decade.
The results of the experiment don’t “leave a lot of space for different interpretations,” says Susana Chuva de Sousa Lopes, a developmental biologist at Leiden University Medical Center in the Netherlands who served on the PhD dissertation committee of coauthor Sarita Panula but was not involved in the research. It seems, she says, that cells previously identified as ovarian stem cells are in fact perivascular cells, which support blood vessel structure and help regulate blood flow.
But the discoverers of ovarian stem cells in adult mammals and other proponents of the cells’ existence are not convinced, citing methodological weaknesses of the new study.
Until relatively recently, scientific consensus was that a female mammal’s oocyte pool is fixed at birth. Adult ovaries, it was assumed, are simply unable to generate new eggs. But in 2004, Northeastern University reproductive biologist Jonathan Tilly and colleagues published findings that appeared to upend this understanding of oocyctes by presenting evidence of ovarian stem cells in adult mice.
A few years later, scientists in China claimed to have also found such germ line stem cells in the ovaries of adult mice, and showed that these cells could differentiate into functional eggs that gave rise to viable mouse pups. And in 2012, Tilly’s group reported the existence of germ cells in samples of human ovarian tissue, claiming that these cells could similarly generate oocytes in vitro and in vivo when injected into mice.
These findings generated a lot of publicity because they suggested that human fertility wasn’t fixed after all. But the data “has always been criticized,” says Fredrik Lanner, an embryonic stem cell researcher at the Karolinska Institute and a coauthor on the newly published study that failed to find such stem cells.
We quite feel certain to say that in the human adult ovary in this cortex region, there is no cell that would be the oogonial stem cell.—Pauliina Damdimopoulou, Karolinska Institute
While some groups have been able to reproduce the results, others have tried and failed. Debates have erupted over methods, techniques, and protocols, and Tilly and his colleagues have published lengthy replies to those who have challenged their work. Today, the field is more or less divided into two camps regarding the existence of ovarian stem cells, says Chuva de Sousa Lopes.
To try to get to the bottom of the issue, Lanner and his collaborators harvested high-quality ovarian tissue samples from 21 healthy patients of reproductive age and isolated the ovarian cortex, the outer layer of the ovary where researchers claim to have found the elusive stem cells. The team used enzymes to break down the ovarian tissues, yielding 24,000 individual cells in total, then performed single-cell transcriptome and cell surface marker profiling, revealing six main cell types: oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. None of the single-cell profiles matched those of reported ovarian stem cells.
When Lanner and colleagues stained the cells with an antibody against DDX4, a germ cell marker that is reported to select for oogonial stem cells, they found that they had instead isolated perivascular cells. The team then stained intact ovarian tissue and saw that the antibody similarly identifies perivascular cells. A comparison of the 24,000 cells to existing transcriptome data from both human fetal ovaries and the ovarian medulla, the inner region of the ovary, also failed to reveal any oogonial stem cells.
“We quite feel certain to say that in the human adult ovary in this cortex region, there is no cell that would be the oogonial stem cell,” says coauthor Pauliina Damdimopoulou, a cell biologist at the Karolinska Institute. She believes that other researchers have succeeded in using the DDX4 isolation technique to select and culture cells, but that what they have found are in fact perivascular cells and not oogonial stem cells.
This study “again highlights that the DDX4 isolation technique is not something that can be used to isolate oogonial stem cells,” University of Adelaide cell biologist Keith Jones, who was not involved in the work but coauthored a 2016 paper suggesting that the same antibody does not isolate DDX4 positive cells, writes in an email to The Scientist. “It brings into question the existence of such stem cells, and leads us back to the dogma that prevailed previously in the field—the adult ovary does not contain oogonial stem cells.”
Damdimopoulou also notes that she and her colleagues found that small, mature oocytes can slip through the filtration process, and when cultured, may appear as if they had been generated from stem cells. “We think [the oocytes] were there all along from the beginning,” she says. The formation of new vasculature by perivascular cells surrounding these oocytes, Chuva de Sousa Lopes suspects, could trigger dormant egg cells to become active and then mature, which might explain the results published by other labs.
Perivascular cells don’t undergo meiosis, perivascular cells don’t express meiotic genes, perivascular cells don’t express germ cell genes.—Jonathan Tilly, Northeastern University
Others are not ready to give up on the idea of ovarian stem cells just yet. Deepa Bhartiya, a stem cell biologist at the National Institute for Research in Reproductive Health in India who was not involved with the research, has been working with ovarian stem cells since 2010 and says that “they can be easily detected.” Research with sheep ovarian tissues has shown that “simple scraping of [the] ovary surface can show the presence of stem cells amongst the ovary surface epithelial cells,” she writes in an email to The Scientist. The problem with the new study out of Sweden, Bhartiya says, is the speed at which the researchers spun their cells—much too slow to isolate the stem cells, which due to their small size do not pellet down at lower speeds and are therefore unknowingly discarded. Bhartiya writes that the study used novel techniques, but revealed nothing new: “if sample preparation is not proper—one will get negative data.”
Tilly argues that there are numerous methodological problems with the study. He says that at this point four independent groups have reported on the existence of human oogonial stem cells, showing that the cells can generate new oocytes in both somatic ovarian tissue and outside the body in culture, and that they can undergo complete meiosis, a germ cell-specific event. “Perivascular cells don’t undergo meiosis, perivascular cells don’t express meiotic genes, perivascular cells don’t express germ cell genes,” he says.
What the field really needs, says Chuva de Sousa Lopes, is more communication among researchers. “The scientists that claim there are stem cells in the ovary and the scientists that are against that are somehow not really talking to each other,” she says. “I wish there would be more open dialogue, because sooner or later all these populations [of cells] will be clarified . . . and things will be more clear.”
M. Wagner et al., “Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells,” Nat Commun, doi:10.1038/s41467-020-14936-3, 2020.
Amy Schleunes is an intern at The Scientist. Email her at email@example.com.
Clarification (March 13): This story has been updated to specify that Jonathan Tilly of Northeastern University referred to reports of human oogonial stem cells by four independent groups.