The grand challenge

First came the egg-in this case, an ordinary mouse egg.

Scientists removed the egg's nucleus and replaced it with the nucleus from a skin cell of a mouse suffering a genetic immune deficiency. Next they manipulated the egg to develop into a blastocyst, a hollow ball holding the embryonic stem cells with the potential to become any cell in the body. The researchers then plucked out the stem cells, corrected the genetic defect, and used the cells to treat the immune deficiency. And the mouse was partially cured.

Announced three years ago by the labs of Whitehead Member Rudolf Jaenisch and then Whitehead Fellow George Daley, this was the first successful "proof of principle" that somatic cell nuclear transfer actually could help to cure disease.

But this fall as Jaenisch opens the Whitehead Human Embryonic Stem Cell Facility, he won't be working directly toward replicating this achievement in humans. Instead, the first order of business is to study the cells' basic biology. Deep and tough problems must be solved long before embryonic stem cells can become useful clinically, Jaenisch says.

Growing pains

One obstacle is simply in learning how to work with these enigmatic cells.

In 1998, James Thomson at the University of Wisconsin-Madison launched the field by deriving the first human embryonic stem cells using embryos from in vitro fertilization clinics. Despite seven years of experience and the creation of more than 100 stem cell lines worldwide, scientists still do not know the best methods for deriving and growing them.

Biologists have encountered severe problems growing the earliest stem cell lines, including most of the lines eligible for federal research funding under President Bush's 2001 decree. "Those lines are very hard to grow and very hard to keep pristine," says Irving Weissman, director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine. He notes that many of the first lines derived now show genetic abnormalities that limit their utility.

"We are far from knowing what the optimal conditions are for culturing human embryonic stem cells," agrees Kevin Eggan, assistant professor of molecular and cellular biology at Harvard University. "So when we derive them and culture them, we are almost certainly doing things that mess them up."

What makes a stem cell?

Even as they struggle to grow human embryonic stem cells, biologists also face basic questions about how they work. The most fundamental of these is "stemness"-what makes a stem cell a stem cell.

Scientists are just beginning to work out the internal programs and external cues that give the cells their unique ability to become any other type of cell, that maintain them in this state, and that allow them to self-renew, almost indefinitely.

Whitehead Member Richard Young, collaborating with Douglas Melton, co-director of the Harvard Stem Cell Institute, is mapping the internal mechanisms and gene/protein interactions involved in stemness.

To uncover the proteins that control gene expression in stem cells, the team is employing techniques that Young helped develop to study the regulatory pathways of baker's yeast. "There is a perception that human embryonic stem cells may be useful for regenerative medicine," Young says. "But before we get there, many of us believe that we have to understand these pathways a little better."

Like Young, Princeton's Ihor Lemischka is tapping systems biology techniques, such as gene chips that represent the total genome, to identify all the genes that are active in embryonic stem cells but not in more mature cells. Austin Smith, director of the Institute for Stem Cell Research at the University of Edinburgh, has taken more classical genetic and biochemical approaches to working out the molecular pathways for self-renewal, creating mutations in specific genes or exposing cells to different substances to test their effects.

Getting with the program

Understanding these pathways will help researchers with another quest of stem cell biology: deciphering how transferring the nucleus of an adult cell into an egg effectively reprograms that nucleus, resetting its genes to the beginning of embryonic development.

Scientists have employed this technique to clone animals, starting with Dolly the sheep in 1997. In 2002, Hwang Woo Suk and colleagues at Seoul National University used it to derive a stem cell line that matched a specific patient. "Understanding reprogramming at a basic level could have a major impact on the whole field," says Leonard Zon, professor of pediatrics at Harvard Medical School and past president of the International Society for Stem Cell Research.

Knowing how the process works could reward stem cell scientists doubly. It would give them a way to derive stem cells without using embryos (thus avoiding that ethical controversy). It also would provide a means of cloning stem cells customized to treat individual patients without the need for more egg cells.

"For everyone's idealized vision of personal cell replacement therapy to come true, we will need something like this," says Eggan.

Eggan gained great proficiency with nuclear transplantation technology as a graduate student in Jaenisch's lab. In his Harvard lab, he investigates whether embryonic stem cells can accomplish the same sort of reprogramming that an egg does.

As a first step to test the possibility, Eggan's group fused a human embryonic stem cell to a skin cell and watched to see whether the hybrid cell would act like a stem cell; it did.

The hybrids have limited usefulness, since they contain double the usual amount of DNA. But the experiment did demonstrate that embryonic stem cells might contain the same unknown substances that stimulate reprogramming in egg cells.

Decoding the genetic disease

Even in its current state, nuclear transfer technology offers biologists a unique tool for studying genetic disease. Most dramatically, in June Hwang and his colleagues used it to create embryonic stem cell lines from patients with type 1 diabetes and a genetic immune deficiency.

The technique, Weissman says, will enable researchers to figure out exactly how such diseases develop-discoveries they may not be able to make any other way. "That's why it's such a big platform technology," he says. "You can make a cell line with a genetic disease, you can study it in a test tube, you can send it around to everybody who's interested, and you can also put it into an animal model where there's a chance that the disease will happen." Weissman hopes to eventually produce such stem cell lines at Stanford with backing from California's Institute for Regenerative Medicine.

Eggan has requested permission from Harvard to derive human embryonic stem cell lines from patients with Parkinson's and Lou Gehrig's diseases using private funding.

Training issues

Biologists have been trying to create particular cells and tissues from human embryonic stem cells since their discovery. Without an understanding of underlying developmental pathways, progress has been slow.

"This question of how to make the embryonic stem cells into tissues is a basic-science question," says Zon, who is working at Children's Hospital in Boston with George Daley to develop methods for directing embryonic stem cells to become blood-forming cells. Such projects obviously play into therapeutic hopes for embryonic stem cells. Zon and Daley, for example, want to cure diseases such as sickle cell anemia and immunodeficiency disorders.

But existing techniques for differentiating embryonic stem cells into specific cell types have proved inefficient, leading to mixtures of cells at different stages of development.

"If you read the papers, you'll see people say it looks like three or four percent of the cells can develop into [heart cells], or 80 percent seem to be motor neurons," says John Gearhart, director of stem cell biology at the Johns Hopkins Institute for Cell Engineering. "We see these numbers all over the place, and invariably this tells you how inefficient the system is."

Crossing presidential lines

As they work to answer these key questions, each of these researchers either uses or plans to use non-presidential embryonic stem cell lines.

While the presidential lines have already helped biologists with some basic research, all of them were derived using mouse cells or components of animal blood to feed the embryonic stem cells.

This not only makes the lines unsuitable for clinical use but also complicates basic science. Researchers have recently begun deriving human embryonic stem cell lines without using any animal tissue, lines that Gearhart thinks will give a big boost to the field. "We would certainly benefit enormously by new lines that don't require mouse feeder layers," he says. "I can't express how much of a pain they are."

Without mouse feeder cells, it should be easier for scientists to investigate the mechanisms that keep stem cells undifferentiated and control their development, he adds. And when biologists ultimately succeed in producing specific cell types such as neurons or muscle cells from such animal-free embryonic stem cell lines, those cells might be moved more directly into clinical trials.

The presidential lines "aren't sufficient," Jaenisch declares flatly. "They are not behaving the way they should be anymore."

So at least for now, Whitehead's new lab must be funded entirely with private money. But private funding cannot completely fill the gap for U.S. researchers. "The National Institutes of Health are the key funding source," says Jaenisch. "If they're not there, it is a major, major problem."

And even when researchers manage to obtain private backing, the federal funding ban has made it much more difficult for those wishing to work with non-approved lines.

As they wait for public opinion and political will to match their need, U.S. researchers work with what state and private money they can raise. As the field races ahead worldwide, Whitehead scientists aim to stay in the vanguard. "Embryonic stem cells hold enormous potential," says Jaenisch. "We have to be sure that we can realize that potential."

Four big questions

Here's a sampling of the human embryonic stem cell issues being tackled by Whitehead scientists:

1. What makes an embryonic stem cell a stem cell? Using microarray technology developed at the Institute, Whitehead Member Richard Young, collaborating with Rudolf Jaenisch and others, has discovered the first layer of circuitry that enables such cells to be pluripotent, suppressing entire networks of genes essential for later development.

2. How can we manipulate embryonic stem cells? Jaenisch and Young are exploring ways to influence key genes and proteins, as well as tampering with their regulatory circuitry. Understanding these processes is a prerequisite for systematically coaxing embryonic stem cells into forming particular cell types.

3. How does an egg reprogram the genome in somatic cell nuclear transfer? The Jaenisch lab is investigating the exact biochemical processes that the egg uses to reactivate the donor nucleus during somatic cell nuclear transfer. The long-term goal is to turn a mature cell into an embryonic stem cell without requiring an egg.

4. How can we make adult stem cells out of embryonic stem cells? In this area, embryonic stem cell researchers and adult stem cell researchers need to work together. The Lodish lab is working to create adult blood cells out of embryonic stem cells, drawing on the expertise of Jaenisch, Young and Whitehead Fellow Fernando Camargo.

Written by Erika Jonietz.

This article first appeared in the Fall 2005 issue of Paradigm magazine.