A stem cell has been described simply as having a unique capacity to renew itself and give rise to specialized cell types. It is this distinction that has made stem cell research so alluring to medical researchers. It ushers in an entirely new weapon in the war against inherited disease. Ralph Brinster, VMD, PhD, is a chief architect.
In an interview with DVM Newsmagazine, Brinster, the Richard King Mellon Professor of Reproductive Physiology at the University of Pennsylvania talked about his National Institutes of Health-funded research in identifying the growth factors essential to allow spermatogonial stems cells to exist in culture, which is considered a major step in an ultimate quest to understand infertility in humans and agriculture.
The resulting benefits from Brinster's work with colleagues Hiroshi Kubota and Mary R. Avarbock will have "profound consequences to future fertility therapies and offer a source of stem cells that will make it possible to modify genes from males before they are passed on to the next generation," Brinster says.
"In 1998, I published the first paper on culture, but the cells eventually died out over three months. So, we spent a lot of time in the laboratory studying cultures."
Eventually, Kubota went into the laboratory armed with information about enriching stem cells and other empirical observations. The result? Kubota developed a very rich population of stem cells. He came up with a serum-free media. The last ingredient to the successful culture was the use of simple feeder cells known as STOs.
"So now the cells can be cultured, and I believe they will proliferate for a very long time, perhaps forever," Brinster says. "We now already have a way that we can freeze these cells, so basically it makes any male germline immortal because you can freeze it, put it back into a recipient and make sperm. That is very different than freezing sperm. You are limited with all of the offerings a male can make. If you freeze 1 million spermatozoa, you have a million spermatozoa."
Divide and conquer Adult stem cells "are capable of making identical copies of themselves for the lifetime of the organism. Adult stem cells usually divide to generate progenitor or precursor cells, which differentiate or develop into "mature" cell types that have characteristic shapes and specialized functions. (Stem Cells: Scientific Progress and Future Directions; Winslow, 2001.)
"With the ability to culture these cells, you can do a lot of experiments, biology and just manipulate them in any way you want. More specifically you can add or delete genes in a stem cell. It's just a matter of applying already known techniques to introduce these genes or go make a mutant."
The implications? "For research, this opens up a wonderfully robust diagnostic system for analyzing the function of individual genes. For medicine, it opens up a new chapter in fertility medicine," he says.
Brinster also believes these results will also be applicable to other species, although the research has primarily focused on mice.
While the female germ cell stops dividing before birth, the spermatogonial stem cells continue to divide throughout life. So, it's possible to modify the male germ line between generations by manipulating the spermatogonial stem cells in culture.
Theoretically, it will be possible to harvest the male spermatogonic stem cells, correct the gene in culture and implant the stem cells back into the male to produce normal sperm.
Brinster also theorizes that it might be possible to convert spermatogonial stem cells to totipotent cells, capable of becoming almost any other cell type and similar to embryonic stem cells.
In terms of infertility problems, the research may offer the most advantages, Brinster says. For example, males who are treated with chemotherapy for malignancies, especially for boys in which there isn't sperm to save, may benefit. "In order to save the germline of that individual, you might take a testis biopsy, grow it and expand it in vitro, and freeze the cells to eventually re-implant them back into the male after the chemotherapy. In some infertility in humans and domestic animals there can be stem cells present even in those animal's deemed infertile. It might be possible to expand those stem cells and use them in a recipient to make sperm.
Brinster and other laboratories are working on a system for in vitro spermatogenesis, thereby eliminating a recipient that is difficult to manage.
Applications for the use of stem cells seem as varied as the genome itself. Just looking at it from a reproductive vantage point, the potential is enormous in creating healthier animals more resistant to disease. The serum-free culture media (stem cells hate serum) is paving the way.
Scientific slight of hand Here's how it worked: The research team identified a single growth factor: glial cell line-derived neurotrophic factor was vital for promoting a signal-pathway that allowed the cells to multiply in culture. GDNF, originally identified as a survival factor for neurons in the brain, was also found to be excreted by the Sertoli cells that surround and support the spermatogonial stem cells in the testes. Once added to the culture, GDNF caused the stem cells to form dense clusters and proliferate continuously.
The researchers then used a gene marker, GFP, in the cultured stem cells to identify the cells before transplantation back to infertile mice. These mice then produced offspring. The expression of the GFP gene made the mice glow green.
Editor's Note: Information for this story also provided by Greg Lester, University of Pennsylvania.