For the first time, researchers have produced healthy mice offspring using sperm derived from precursor cells generated in vitro. The achievement was made possible through the development of a highly efficient protocol for generating the sperm precursors from mouse embryonic stem cells.
The research, which appears online today (August 4) in Cell, could open doors to better characterizing the poorly understood process of germ cell development, as well as suggest possible avenues for the development of fertility treatments.
“This is a fascinating story, a technical tour-de-force,” said George Daley, a stem cell biologist at Children’s Hospital in Boston, who was not involved in the study. “This study establishes a major new platform for investigating sperm development, which may have implications for the development of male contraceptives and insights into fertility in general.”
Although germ cells differentiate to form every cell type in the body and are responsible for passing both the genetic and epigenetic information of a sexual organism from generation to generation, little is known about how they arise. The direct precursors to germ cells, known as primordial germ cells, appear early on during embryonic development (around the first week after fertilization in mice). But because they exist in very small numbers—between 100 and 1,000—and are very quick to differentiate, they have remained notoriously hard to isolate and characterize.
Several groups have successfully generated primordial germ cells in vitro from embryonic and induced pluripotent stem cells, but the resulting culture of differentiated cells usually contained only a small percent of primordial germ cells whose function was never thoroughly assessed. In 2007, scientists created a new kind of stem cell, derived from an early embryonic tissue known as the epiblast, which generates the three germ layers of the embryo. Because epiblast cells are the direct precursors of the primordial germ cells, researchers were hopeful that the novel cell line, called epiblast stem cells (EpiSCs), might provide a more efficient in vitro source for germ cells. Unfortunately, EpiSCs have proved just as inefficient as a starting point as the other types of stem cells.
When Mitinori Saitou, a stem cell biologist at Kyoto University in Japan who led the current study, and his colleagues compared the global gene expression of the epiblast stem cells with the expression in actual epiblast cells from a mouse embryo, they found that they were, in fact, quite different. This led them to search for a different starting cell line—one that could better recapitulate the population of epiblast cells that give rise to primordial germ cells in an embryo. Specifically, they decided to take a step backward and culture the more primitive embryonic stem cells (ESCs), which are derived even earlier in embryonic development.
Using a specific cocktail of Activin A (ActA), a regulator of cell differentiation and proliferation, basic fibroblast growth factor (bFGF), and knockout serum replacement (KSR) as a supplement, the researchers were able to generate a uniform population of epiblast-like cells, with a gene expression profile comparable to epiblasts, in just 2 days. From there, the researchers used a protocol they had established a couple of years ago to differentiate the epiblast-like cells into a primordial germ cell-like state. Finally, using several novel molecular markers, the researchers were able to select the cells that were likely to have the ability to further differentiate into the precursors of sperm.
To test the functionality of their final cell population, the researchers injected the primordial germ cells into the testes of mutant mice that lacked germ cells, and were thus unable to produce sperm of their own. After 8 to 10 weeks, they removed the testes, and to their delight, found evidence of spermatogenesis: thickening seminiferous tubes that were producing sperm. Finally, through artificial insemination of those sperm into normal females, the researchers were able to produce seemingly healthy baby mice. “So regarding the function of the sperm, I think they are very normal,” Saitou said.
“Part of the excitement of this new study is that it now allows a much greater number of primordial germ cells to be generated in culture,” said Peter Rugg-Gunn, a stem cell biologist at The Babraham Institute, Cambridge, UK. “This will really open up the possibility of a lot of additional assays that will allow the primordial germ cells to be characterized in much greater detail than was able to be achieved using previous differentiation protocols.”
An aspect of germ cell biology that the new protocol may inform, for example, is the mysterious process of epigenetic reprogramming, Saitou said. “Only in the germ cells there is this very interesting editing of epigenetic information that happens to make a totipotent zygote.”
Other next steps for the researchers are to repeat this experiment with the female germ cell line as well as extend the protocol to produce the spermatogonial stem cells that give rise to sperm in vitro.
K. Hayashi, et. al., “Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells,” Cell, 146: 1-14, 2011.