Three colonies of GFP-labeled undifferentiated human embryonic stem cells (cell line UC06) grown by NHLBI's Tom Cimato, with technician Jeanette Beers of NHGRI and Kye-Yoon Park of the NIH Stem Cell Characterization Facility, who introduced the green protein into the cells

Pam Robey describes it as an art, coaxing stem cells in a culture to behave the way nature would have them behave in the body.

"A lot is in the way it feels," she says. "There are nuances you can’t quite put into words."

Robey, chief of the Craniofacial and Skeletal Diseases Branch, NIDCR, is conducting a comparative stem-cell study to understand the genes involved in pluripotency and their effect on disease.

With expertise primarily in postnatal cells, she and her staff three years ago turned to what was then the newly established NIH Stem Cell Unit for a headlong plunge into embryonic stem cells. It was the best way she knew to get to the root of the problem.

The end goal—years away, she readily acknowledges—is to grow tissue to cure diseases such as fibrous dysplasia, a bone development abnormality. But along the way, she is learning the essence of how disease develops.

Not that the path is so straightforward. As anyone who has wrestled with embryonic stem cells would testify, they need more than a dash of Miracle-Gro.

To her frustration, Robey found early on that cultures would be growing fine one day, then die the next for no apparent reason. Or they would spontaneously differentiate. "These cells are just plain hard," she says. "There’s nothing easy about them."

And so she knocked on the door of what is now known as the Stem Cell Characterization Facility, still one of the best-kept secrets on the NIH campus. Through hands-on training both in their own lab and at the facility, Robey's colleagues, biologist Joanne Shi and staff scientist Sergei Kuznetsov, became adept cell cultivators.

Employing a mix of scientific rigor and artful pipetting, they can now grow the embryonic stem-cell line known as HSF6 and ward off spontaneous or otherwise unwanted differentiation with near regularity. And the group is now off and running on its research project.

It’s all in the wrist: Exercising what NIDCR lab chief Pam Robey calls the "art form" of pipetting when it comes to growing embryonic stem cells, staff scientist Sergei Kuznetsov adds growth fluid to stem cells	The face behind the wrist: Sergei Kuznetsov

Learning Curves

Nearly three years into the stem-cell- growing business, the facility is renewing its effort to reach out. Having cultured 17 of the 21 federally approved human embryonic stem-cell lines—with great success in karyotyping them and establishing growth protocols—the facility wants to place more emphasis on training intramural scientists.

"We are developing a world-class facility at NIH," says Ron McKay, the NINDS investigator who runs it. He deems the facility one of the best laboratories in the world to develop expertise in human embryonic stem-cell science. "The whole technology is on the move. We are inviting people to participate."

Along the lines of the adage about teaching a person to fish, McKay sees the facility as teaching the skills of how to grow embryonic stem cells, and he is planning a series of special seminars and training sessions this year. Without some training, he notes, one can waste a lot of time trying to grow undifferentiated embryonic stem cells.

In the stem-cell facility, all the cells are grown under identical conditions, and they are mostly normal, McKay says. His group has crafted a relatively reliable experimental system—essential to accomplishing anything with the cells.

The stem cell facility crew: (left to right): Kye-Yoon Park, Barbara Mallon, Becky Hamilton, and Kevin Chen

Growth Curves

Barbara Mallon, a scientist at the facility since its incarnation as the NIH Stem Cell Unit in 2004, speaks of stem cells as invigorating the very core of basic cellular research.

Yes, the science can be intimidating, and the methods daunting, Mallon observes, but the reward will be unprecedented advances in biomedical science. The facility has much to offer in terms of handholding and lesson sharing, she says, but sometimes scientists unfamiliar with its purpose come with false expectations. 

"We want people to understand that we’re here but also what we do, and what we can and can’t do," Mallon said. "Some people think we’ll be able to supply them with cells. That’s not really our role."

That is, her group cannot make copies of undifferentiated cell lines for scientists to take back to their own labs. Doing so would be analogous to bootlegging a DVD and is forbidden by the Material Transfer Agreement set by the suppliers. NIH possesses the stem-cell lines but does not own them. The owners, companies such as WiCell Research Institute in Madison, Wisc., stipulate what can be done with the cell line.

What the Stem Cell Characterization Facility can do is offer quality training and support simply not available elsewhere. For visiting NIH scientists there is the opportunity to use the facility’s cells and equipment, housed in Building 37. Mallon and her colleagues—Kye-Yoon Park, Kevin Chen, and Becky Hamilton—also visit other NIH labs to assist.

There are other places around the country that offer training, for a steep price; but these are short, one-shot deals, Mallon said, and scientists returning to their labs often must face myriad culturing problems on their own.

For example, Mallon says, "people can grow cells, but they can be growing junk and not realize it. The markers that are used may not be sensitive [enough] to detect the fact that your cells are going downhill." 

Her group has been there and done that with trial and error and a little more error. They have established standards for growth, continue to make growth easier and better, and know how to monitor the health of the cultures. These were hard lessons to learn that they can now pass on.

"Checking his garden" is the way NHLBI clinical fellow Tom Cimato describes how he monitors the progress of his stem-cell colonies	In full view: Tom Cimato, with the picture behind him of the 21-cell stem-cell colony he viewed under the microscope

Biology 101

Stem cells, says McKay, are at the root of diseases such as cancers and neurological disorders. "How do you figure out the relationship between mutation and altered phenotype," McKay asks. "That's why this is important."

"Stem-cell biology is part of the continuum of understanding human development, growth, and differentiation," says NHLBI Director Elizabeth Nabel, who is working with NHLBI’s Tom Cimato, a clinical fellow, to grow cardiac tissue.

Nabel is among the IC leaders who encourage their staff to become more active in and stay abreast of this type of research. "I think stem cells provide the perfect niche of applying many of those principals of basic biological processes to clinical medicine. We’re very keen in supporting that translational work," she says.

NIDCR’s Pam Robey discussing fibrous dysplasia in 2002

NHLBI’s Elizabeth Nabel and NINDS’ Ron McKay at a panel discussion on adult stem cell research at NIH in 2004

Indeed, Cimato, who occupies one of the facility’s two visiting scientist spots, was attracted by the "Biology 101" quality of stem-cell research. He says that heart disease is, in essence, a stem-cell disease. Some toxin, perhaps nicotine or a fatty acid, he muses, disrupts the routine process of replacing endothelium and vascular cells. 

Cimato is tracking down the cardiac stem cell, asking, "how does it grow, how does it differentiate, how many steps does it go through in creating new endothelium?"

And he’s closing in on some answers. He has successfully nursed an embryonic stem cell through the stages of differentiating into endothelium, one step away from cardiovascular tissue. To do so, he has had to learn how to inhibit all other kinds of differentiation while simultaneously enticing the the cells down the path to endothelium, a two-year laboratory effort. 

Like others, he calls upon a certain intuition to grow these cells, an intuition, he observes, that would have been difficult to develop without the help of the stem-cell facility.

Cimato sees embryonic stem-cell science as enabling unambiguous insight into disease development.

Robey, taking a different path of differentiation, has arrived at a similar conclusion. She calls fibrous dysplasia a skeletal stem-cell disease. 

"Any change in a skeletal stem cell’s metabolism, either by mutation or change in the microevent, will result in a skeletal disorder," she says. Yet how that disorder manifests depends on the stem cell. She stumbled upon this in her work on fibrous dysplasia, she recalls. The same mutation caused lytic bone growth below the neck yet sclerotic growth above the neck, a phenomenon that traces back to the cell origin, either mesoderm or neuroectoderm. 

Robey has traced the three features of fibrous dysplasia—patches of café au lait skin, precocious puberty, and dysplasia—to the three germ layers.

Embryonic Technology

The Stem Cell Characterization’s Facility’s services are different from, say, DNA sequencing, where the technology is mature enough to set a price for a given product to be produced in a given time period. "We’re not there yet," McKay says. In other words, fees vary.

He anticipates that interactions with the NIH community will increase as the power of the technology grows.

Nabel notes that the field is young: "We are still in our early years in terms of understanding the biology of stem cells—understanding how stem cells grow and differentiate, what markers are expressed at different points of differentiation. A lot of that is simply descriptive discovery work that needs to be done. Once we have a better handle on the cells per se, then I think the opportunities for application will be enormous."

For more information on stem cell facility services and fees, contact Barbara Mallon; For more information on stem cells, visit this website.