RESEARCH | A valuable racehorse siring generations of potential champions long after his death. An infinite supply of stem cells spawning neurons for people with spinal-cord injuries. A healthy child born to a father who carries a genetic defect.
These dreams have been brought one giant step closer to reality by the recent work of Dr. Ralph L. Brinster V’60 Gr’64—the Richard King Mellon Professor of Reproductive Physiology and arguably the world’s premier contributor to the field of germ-line genetic modification—and several colleagues at the School of Veterinary Medicine.
Brinster’s group developed a groundbreaking technique for growing spermatogonial stem cells (SSCs)—the continually self-renewing cells that give rise to sperm—in culture, perhaps indefinitely. The result is a virtually bottomless repository of male gametes-in-waiting, primed for genetic manipulation.
“The ability to harvest and culture spermatogonial stem cells offers the potential to improve animal health and productivity, and to treat human infertility,” says Brinster.
In the study—“Growth Factors Essential for Self-renewal and Expansion of Mouse Spermatogonial Stem Cells,” published last November in the Proceedings of the National Academy of Sciences—the newly cultured stem cells were injected into the testicles of sterile male mice.
These transferred SSCs, which carried the GFP (green fluorescent protein) marker, initiated normal spermatogenesis—sperm production—in the recipient mice. The proof: These congenitally infertile mice sired mouse pups—not just any old mouse pups, but a luminescent version that fluoresced the same brilliant apple-green hue as the SSCs from which they arose.
Key to the project’s success was identifying a central signaling process that spurs the SSCs to continually regenerate, essentially becoming immortal. The signaling agent, glial cell line-derived neurotrophic factor (GDNF), is a natural body chemical that also nurtures neurons in the brain.
GDNF also supports the SSCs in the testes and, when added to Brinster’s serum-free culture—which included fibroblast growth factor—caused the stem cells to form dense clusters and proliferate continuously for six months-and-counting.
“The most important element of this research is that it identifies the essential factors that support the growth of these cells,” explains Dr. Hiroshi Kubota, a research assistant of cell biology who was the paper’s lead author.
The SSCs, which are sparse in number (one in 3,000 adult testis cells), offer a stark measure of the sexes’ different reproductive potentials. Since SSCs replicate throughout life, they render the male perennially fertile. But the female germ cell—the oocyte—stops dividing before birth, at which time a finite number of eggs are in place. The reproductive lifespan of the female is thus limited.
Much scientific bench-work has focused on using reproductive technologies to manipulate the egg. In the early 1960s, Brinster developed a culture technique for mammalian eggs that set the standard worldwide. Soon after, he began to investigate the metabolic requirements of egg development. With his subsequent exploration of methods for manipulating the genome of eggs in vitro, the field of animal transgenics was born.
In the early 1990s, Brinster turned his attention toward spermatogenesis, which he described as “one of the most complicated, highly organized and efficient processes in the body.” In 1994, he developed a technique for transplanting SSCs from fertile to infertile mice, which then produced sperm. But to grow them in a dish was another hurdle altogether, since the required culture conditions—growth factors in particular—were not yet understood.
On the female side of the equation, scientists were closing in on some answers.
Two years ago, a team of researchers at the veterinary school led by Dr. Hans Scholer, director of Penn’s Center for Animal Transgenesis and Germ Cell Research (and the Marion Dilley and David George Jones Chair and Professor of Reproductive Medicine), became the first to produce eggs from embryonic stem cells, which are uncommitted cells capable of becoming just about anything [“The Most Amazing Cell,” September/October 2003].
Last year, when Brinster, Kubota, and research associate Mary R. Avarbock, working in Brinster’s lab in the veterinary school, successfully cultured sperm-forming SSCs, they introduced several exciting possibilities. For one, Brinster predicts that within five years, these results—now limited to mice—will be extended to other species, possibly even humans. By providing a renewable source of sperm, SSC culture could be used to enhance male fertility—even for cancer patients, who may be rendered sterile by their treatments.
“It immortalizes the germ line of the male,” says Brinster, who hopes that he and his colleagues can expand these techniques to in vitro spermatogenesis. Instead of implanting SSCs in a recipient, scientists might soon be able to prod them to develop into mature sperm in culture.
“If we can generate functional sperm cells in culture,” Kubota explains, “we can learn in detail each step of differentiation and then develop methods to fix the genetic defects.”
A gene encoding for a disease like Tay-Sachs, for example, might be corrected by manipulating the sperm in vitro. Finally, SSCs can be converted to embryonic stem cells, from which a variety of tissues might one day be grown to help those suffering from diseases like Parkinson’s and kidney failure.
Spermatogonial stem cells link us—literally—to future generations. And the means to culture them may ultimately bridge us to the future of medicine.
—Joan Giresi C’86 V’98