When Science voted stem cell research its 1999 Breakthrough of the Year, the congratulatory article traced the field's origin to the 1981 successful culture of mouse embryonic stem (ES) cells.1,2 But the roots of exploring these multipotential cells go back considerably farther, to a little-mentioned researcher who worked with mice at the Jackson Laboratory in Bar Harbor, Maine.
|Photo: Jackson Laboratory|
One day, he noticed a mouse with a huge scrotum. "We killed it, and looked at the testes, and they had strange things inside," recalls Don Varnum, a long-time technician in the lab. The growth included a mishmash of tissue, including hair and teeth. It was a teratoma, and other mice of strain 129 had them too. Intrigued, Stevens pursued federal funding to explore teratomas, and the support lasted a professional lifetime. "This stuff was extremely interesting, and it sure beat studying cigarette papers!" he recalls from his home in rural Vermont, where he has lived near family since suffering a stroke 10 years ago.
Stevens bred strain 129 to select for the teratoma tendency. Then he developed a serial transfer technique to be able to continuously study the rare cells in some of the growths that rendered them cancerous. When and if a teratoma ran out of these embryonal cells, it would just sit there, sporting its peculiar mix of hair and teeth, cartilage, and tiny tubules. "We just wanted to keep the tumor alive longer so we could study it. But it took a long time to succeed," says Stevens.
By passaging teratomas through many hosts and removing them from time to time and cataloging the tissues, the researchers witnessed all that these cells could become. And the odd tissue struggling for identity wasn't as haphazard as it seemed. When Stevens and Varnum transplanted teratoma bits into the peritoneal cavities, curious growths resembling inside-out embryos formed, called embryoid bodies. The tissues from the teratomas, given signals from the ascites fluid of the peritoneal cavity, seemed to be trying to organize.
The researchers continued to describe the tissues of teratomas and embryoid bodies, supplementing their publications with spectacular atlases of photographs that captured the temporal unfolding of this deranged development.3,4 Although the anatomy was clearly off, both out of place and out of time, Stevens and Varnum noted that the events followed a sequence of sorts. "Roy looked at thousands and thousands of mice. He noticed that for tissues to develop, several tissues must be in contact, so that the cells know whether to become liver or kidney," Varnum recalls.
Although Stevens' and Varnum's work was purely basic research, the events that they so carefully chronicled, and the biochemicals from without and within that fueled the choreography of early development, are what stem cell researchers and tissue engineers are deciphering today. Tracing Teratoma Origins Leads to ES Cells
With a steady supply of teratomas and their tissues identified, Stevens moved on to other questions--where, and when, did development take a wrong turn? And so he began looking backward, seeking the earliest stage when cells in the testes looked different, and then going back a few more days to account for changes not obvious to a human observer. This approach took him to the genital ridge in a 12-day prenatal mouse, which houses primordial germ cells, sperm precursors.
Then in 1970, Stevens made a leap that would profoundly affect stem cell technology a decade later--he noticed that the primordial germ cells that gave rise to teratomas looked a lot like the cells of considerably earlier embryos.5 So he transplanted cells from various stages of early strain 129 embryos, including inner cell mass cells, into testes of adult mice. Some of the early embryo cells gave rise to teratomas! These induced growths looked and acted like spontaneous teratomas, yielding embryoid bodies when transplanted into mouse bellies and displaying an impressive range of tissue types. Stevens called these cells from early strain 129 embryos that could support differentiation "pluripotent embryonic stem cells"--the origin of the term "ES cell." But because these cells could give rise to cancerous as well as normal cells, they became known as embryonal carcinoma, or EC cells.
The rest, as they say, was history. But quite a lot transpired between Stevens' identification of the developmental potentials of primordial germ cells and inner cell mass cells, and the unveiling of the role of the human versions of these cells as ES cells.
First, Beatrice Mintz and Karl Illmensee, from the Institute for Cancer Research in Philadelphia, visited Stevens to learn his techniques and borrow mice; then they demonstrated that ES cells could give rise to organisms, not just teratomas.6 (Their surprise announcement of this feat at a meeting floored Stevens, a story unto itself.) Then Martin Evans at Cambridge University and Gail Martin at the University of California, San Francisco, and their coworkers showed that inner cell masses from normal mice could support development too.1,2 The field detoured into genetically altering mouse ES cells, which evolved into knockout technology with the harnessing of homologous recombination to target the genetic changes.7,8 Then in the late 1980s, Brigid Hogan, a professor of cell biology at the Vanderbilt University School of Medicine in Nashville, with Peter Donovan's group at the National Cancer Institute, reignited interest in primordial germ cells by devising culture methods. These cells became known as embryonic germ (EG) or embryonic stem-like cells.9 A 1997 article in The Scientist interviewed several of these major players in the stem cell story.10
It wasn't until November 1998, with publication of the two human ES cell papers, that the media and public were finally diverted from Dolly sufficiently to notice this much more powerful technology.11,12 Attention centered on the two cell sources, and still does. James Thomson's group at the Wisconsin Regional Primate Research Center used inner cell masses from in vitro fertilization clinic "leftovers," while John Gearhart's group at the Johns Hopkins University School of Medicine used primordial germ cells from aborted fetuses. And the work that led to their success began in a mouse lab nearly half a century ago, with a man with gifted hands and alert eyes who personifies Louis Pasteur's oft-quoted observation that "chance favors only the prepared mind."
Leroy Stevens is truly the unsung hero of stem cell research.
Ricki Lewis is a contributing editor to The Scientist. A longer version of this article will appear in an essay book she is writing for Blackwell Science, scheduled for publication this fall.
1. G.R. Martin, "Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells," Proceedings of the National Academy of Sciences, 78:7634-8, 1981.
2. M. Evans and M.H. Kaufman, "Establishment in culture of pluripotential stem cells from mouse embryos," Nature, 292:154-6, 1981.
3. L.C. Stevens, "Studies on transplantable testicular teratomas of strain 129 mice," Journal of the National Cancer Institute, 20:1257-70, June 1958.
4. L.C. Stevens, "Embryology of testicular teratomas in strain 129 mice," Journal of the National Cancer Institute, 23:1249-94, December 1959.
5. L.C. Stevens, "The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos," Developmental Biology, 21:364-82, February 1970.
6. B. Mintz and K. Illmensee, "Normal genetically mosaic mice produced from malignant teratocarcinoma," Proceedings of the National Academy of Sciences, 72:3585-89, 1975.
7. T. Doetschman et al., "Targetted correction of a mutant HPRT gene in mouse embryonic stem cells," Nature, 330:576-7, Dec. 10, 1987.
8. K.R. Thomas and M.R. Capecchi, "Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells," Cell, 51:503-12, Nov. 6, 1987.
9. Y. Matsui et al., "Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture," Nature, 359:550-1, Oct. 8, 1992.
10. R. Lewis, "Embryonic stem cells debut amid little media attention," The Scientist, 11: 1, Sept. 29, 1997.
11. J. Thomson et al., "Embryonic stem cell lines derived from human blastocysts," Science, 282:1145-7, Nov. 6, 1998.
12. M.J. Shamblott et al., "Derivation of pluripotent stem cells from cultured human primordial germ cells," Proceedings of the National Academy of Sciences, 95:13726-31, November 1998.