Ready, Reset, Go
Rudolf Jaenisch enjoys climbing mountains, rafting rapids, and unraveling the secrets of pluripotency—knowledge that could someday lead to personalized regenerative medicine.
It was a misbehaving virus that drew Rudolf Jaenisch to epigenetics. As a postdoc in Arnold Levine’s lab in Princeton in the early 1970s, Jaenisch was studying the DNA tumor virus SV40. “The virus makes only skin tumors when you inject it into a mouse,” he says. “Not brain tumors or liver tumors. And I wondered why.” Two possible explanations came to mind: either the virus couldn’t infect brain cells, or it could infect them, but was then silenced so that it could not transform them. To figure out which was correct, Jaenisch needed a way to make sure the virus got into cells other than skin. That’s when he stumbled across the classic 1967 paper by developmental geneticist Beatrice Mintz of the Fox Chase Cancer Center. Mintz had managed to combine cells from two different embryos to form mice that were chimeric. “What blew me away was the idea that you can manipulate a mammalian embryo at such an early stage and wind up with a mouse,” says Jaenisch. He saw the technique as a way to introduce SV40 early enough in development to make sure the virus made it into all of the animals’ tissues.
He just had to figure out how to do it. “At that point, nobody had ever injected DNA into embryos,” says Jaenisch. Mintz was skeptical—as was Levine. “Arnie said, ‘You’re nuts. But if you want to try, be my guest. You can use my lab to make the DNA.’” Mintz taught Jaenisch how to isolate and culture embryos. And when the infected mice were born, they looked totally normal. “I thought there would be tumors everywhere,” says Jaenisch. “There was nothing. That was somewhat disillusioning.”
But had the virus actually gotten in? Jaenisch stained tissues with an antibody against a viral protein—and the result was positive. “I was really excited,” says Jaenisch. “For one night. The next day I did the controls and they were all positive, too. So the antibody was bad.” Then he got a job offer at the Salk Institute. There, Jaenisch learned how to radioactively label his viral DNA. And he used that probe to determine that the mice were, indeed, riddled with SV40. “In the brain, in the kidneys, all over the place,” he says. “These were the first transgenic mice,” which Jaenisch described in a 1974 paper in PNAS—although it would be another six years before that term was coined.
“If this experiment had not worked, I would not be in science today,” says Jaenisch, who has devoted himself to understanding how epigenetic changes silenced these viruses in embryonic cells—and how these modifications are implicated in mammalian development and disease. Here he talks about the promise of induced pluripotent stem cells, the uncertain state of embryonic stem-cell research, and his trip to the Vatican.
Just say “no” to a PhD. Jaenisch received his medical degree from the University of Munich in 1967 and contemplated pursuing a doctoral degree in biochemistry. For advice, he and a friend approached the personnel chief of the Bayer company. “We asked him whether a pharmaceutical company like Bayer would find it worthwhile to have people with both an MD and a PhD. He said, ‘No. We want MDs to sell our stuff and chemists to cook it.’ We thought that was a really good answer so we both decided not to do a PhD.”
And the winner is…In the 1980s, Jaenisch started using viral integration to mutate and identify genes—a technique that came to be called insertional mutagenesis. In his first round of mutagenesis, he shut down a gene that appeared to be important during development: mice homozygous for this mutation died in the womb. After a bit of sleuthing, he found that the gene he’d inactivated encoded collagen. “I thought I’d discovered this fancy developmental gene—but it was just collagen. The most boring gene there was! Collagen makes up something like 60 percent of your total protein!” The mice, however, provided a good model for the human ‘brittle bone’ disease, osteogenesis imperfecta. And the virus, Jaenisch discovered, actually silenced the gene by enhancing its methylation. “So it turned out to be really interesting.”
Let’s clone! In 1996, scientists produced Dolly the sheep by transferring the nucleus of a mammary cell into an enucleated egg. Two years later, scientists in Hawaii announced they had successfully cloned mice using a similar procedure. Jaenisch wasted no time learning the technique. “I thought, if you’re interested in epigenetics, nuclear transfer is the most unbiased way to study the process,” he says. “During nuclear transfer, you have to reset the epigenetic state of a somatic cell to something embryonic. I wanted to analyze the mechanism of this reprogramming. So I went straight to Hawaii to talk to these guys.” His affiliation with Whitehead—an institution of which he was a founding member—allowed Jaenisch to move quickly. “I didn’t have any funding for this work and I didn’t have the instruments, but I knew it was important,” he says. “So I went to the director of the institute, Gerry Fink, and told him what I needed. He said, ‘Well, let me think about it for five minutes.’ That was very fortunate. In another setup, we probably could not have made this happen.”
The path to pluripotency. In 2006, Shinya Yamanaka and his colleagues at Kyoto University introduced induced pluripotent stem (iPS) cells to the world. According to their protocol, making stem cells was as easy as introducing a set of four genes into any somatic cell. “Many people just didn’t believe it,” says Jaenisch. “They said it can’t be so simple.” These first-generation iPS cells were not quite the same as embryonic stem cells: they could not produce chimeras, one of the criteria for pluripotency. But Jaenisch got to work. And one year later, three independent groups—led by Yamanaka, Jaenisch, and a former student of Jaenisch—unveiled iPS cells that were in every way identical to embryonic stem cells. “That meant that you could take a somatic cell, reprogram it in a culture dish, and produce a pluripotent cell that could make a normal mouse. It totally electrified the field.”
Pluripotency takes time. “Many people think you put these genes in and instantly switch from a somatic cell to a pluripotent cell. That is not correct. These genes initiate a long process that’s extremely inefficient. This epigenetic reprogramming occurs over many cell divisions. So making iPS cells takes weeks.” The key step, Jaenisch finds, is the reactivation of a set of three regulatory genes: Oct4, Sox2, and Nanog. “They all interact with one another, and they are totally inactive in somatic cells,” says Jaenisch. “So what the reprogramming process has to achieve is reactivation of this regulatory loop. Once you have that, the cells are stably pluripotent.”
Forget fibroblasts. “Probably all labs use embryonic fibroblasts as the somatic ‘donor’ cell because you can harvest and grow them easily,” says Jaenisch. “But I think this donor cell is not a good choice.” Fibroblast preparations can contain a heterogeneous collection of cells, so one can never be sure whether it was a fully differentiated cell that gave rise to the iPS cell—or perhaps a stem cell from the same tissue. To settle the issue once and for all, Jaenisch and colleagues decided to make iPS cells from mature B cells. These cells have undergone rearrangement of their immunoglobulin genes—a permanent change to the genome. And they found that mice made from these B cell–derived iPS cells had the expected markings of the mature immune cells in their DNA. “That was an important experiment,” says Jaenisch of the study published in Cell in 2008. “Because it showed that you could indeed reprogram terminally differentiated cells. Nobody could doubt it.”
• Generated the first transgenic mice and one of the first transgenic animal models of a human disease.
• Determined that DNA methylation silences viral gene expression in early mouse embryos.
• Was among the first labs to perfect the nuclear-transfer technique for generating cloned mice. In 2002, first demonstrated “therapeutic cloning”—the modification and use of stem cells from cloned embryos to treat genetic defects in mice.
• Working with mature B and T cells, demonstrated that the genome of terminally differentiated cells can be reprogrammed to direct the development of an animal.
• Developed techniques for generating induced pluripotent stem cells from animals and humans.
• Demonstrated that induced pluripotent stem cells can be used to treat sickle cell anemia and Parkinson’s disease in animal models of these disorders.
Flawed clones. “In my view, you cannot make normal clones. Dolly looked normal. But after six years they had to kill her because she was so sick. Mice are the same. Most die very early [in development]. A few make it to birth. And the ones that survive look pretty nice for a year. But many die by 15 months. So I would argue that the animals that survive are just less abnormal than ones that die early. With nuclear transfer you never get normal embryos.”
No therapeutic transfer. “Ten years ago, we talked about the potential of nuclear transfer for therapy. But it turns out the technique was of no practical relevance. You would never do it in humans for a number of reasons. First, it’s very inefficient. With mice, that doesn’t matter because we can do hundreds of transfers to get a few mice. But human cloning is another order of magnitude more difficult than in mice. And people can’t even get the eggs to practice [on]. My former student Kevin Eggan, along with his colleagues at Harvard, spent years putting in place a protocol to get volunteer egg donors. They spent a couple hundred thousand dollars just in advertising. And I think they got one or two donors. Kevin’s postdoc, Dieter Egli, who went to Columbia, told me that he got a couple [of] human nuclear transfers going, but they all arrested at the 6- or 8-cell stage. So there’s something we don’t understand going on in human [embryos]. It should work, but we’re not there yet.”
Funding culture. “In Germany there are these Max Planck positions where you get a lifelong job with a lifelong budget, though this is changing. It was great because it allowed you to pursue high-risk, high-return experiments without having to produce immediate results. But it may have encouraged some people to do little for 20 years, because they’d still get the money. In this country, you have to publish, publish, publish. But you can’t get grants unless you have preliminary data. When money is tight, study sections are very risk-averse. So you are under a constant pressure to produce, but you can’t do something risky. It would be great to have some wealthy donor say, ‘I trust you, here’s $20 million.’”
Stem cells in limbo. “Right now, things are worse than they were under Bush.” A former MIT faculty member, James Sherley, has filed a lawsuit to reverse Obama’s 2009 ruling that allows use of federal funds for work on embryonic stem cells. “He says you don’t need embryonic stem cells, because you can do everything with adult stem cells—which is his science. So his main argument is that he can’t get grants, because the grants go to people working with embryonic stem cells. And that that is discrimination.” A federal appeals court judge agreed with him. “That was in August,” says Jaenisch. “So any grant that was submitted with the name ‘human embryonic stem cells’ was no longer discussed. People who have existing grants on embryonic stem cells—this includes all embryonic stem cell lines, the Bush lines, anything—you can’t work with them anymore. It’s very weird. So now it’s much worse than under Bush. Under Bush at least we had lines to work with. The court decision is under appeal and the case may end up in the Supreme Court. And with this Court, I don’t know how it will come out.”
It may be the field’s dirty little secret: human embryonic stem (ES) cells do not have the same capabilities as mouse ES cells. “You can’t clone them. You can’t use them for gene targeting. They don’t make chimeras. They are very different from mouse embryonic stem cells, which can do everything,” says Jaenisch.
The problem? The culture conditions developed for purifying mouse ES cells just don’t include the factors that human ES cells need to remain in a fully pluripotent state. Now, Jaenisch and team have come up with an improved recipe for making mouse-like human ES cells. First, they’ve attached the reprogramming genes to a promoter that allows them to be switched on in the presence of the antibiotic doxycycline. Then, by incubating conventional human ES cells with doxycycline—along with other molecules that stabilize the naive pluripotent state, such as inhibitors of glycogen synthase kinase-3β and a cytokine called LIF (leukemia inhibitory factor)—they’ve produced stable human ES cells that behave more like those from mice (PNAS, 107:9222-27, 2010).
Greetings. “When I came to Princeton from Germany for my postdoc, that first morning colleagues asked me, ‘How are you?’ The second morning, they asked again. I thought I had to give a different, more sophisticated answer. I was totally confused. I didn’t know this was just what you say and that nobody expects a real reply.”
You are what you eat. “Your diet affects your genome—that’s clear,” says Jaenisch. So, what does he eat? “Mostly fish and vegetables. Not a lot of meat. In the morning I eat fruit and at lunch I eat salad because I know what’s in it.” And for a treat: “Frozen blueberries with rum. And maybe a bit of sugar or nuts on top.”
Tell it to His Holiness. In 2003, Jaenisch attended a conference on stem-cell technologies sponsored by the Pontifical Academy of Sciences. “We thought that maybe we would have some interesting discussions with some very clever Jesuits,” he says. “But they had already prepared a statement saying ‘We’re totally against this.’ So there was no debate. But we stayed at the Vatican, had a private showing of the Sistine Chapel, and shook hands with the Pope. So we had a good time.”
Into the wild. “I like to go to really remote places,” says Jaenisch. He’s weathered snowstorms on glaciers, rafted down the rapids of the Colorado River (four times), and kayaked through Alaska—where he spent nights up a tree avoiding troublesome bears. “I like a challenge,” he says.