The Burroughs Wellcome Fund Career Awards in the Biomedical Sciences (now reconfigured as the Career Awards for Medical Scientists) were designed to help young investigators bridge the career transition between their postdoctoral training and their early faculty positions. Amy Wagers, Ph.D., who is emerging as a leading researcher in stem cell biology, exemplifies the value of providing support at that critical juncture in a young scientist’s career.
Amy received her BWF Career Award—$500,000 over five years—in 2003, when she was still working as a postdoctoral fellow in Dr. Irving Weissman’s laboratory at the Stanford University School of Medicine. In 2004, she became an assistant professor of pathology at Harvard Medical School, a principal investigator in the Section on Developmental and Stem Cell Biology at the Joslin Diabetes Center, and a principal faculty member at the Harvard Stem Cell Institute. As she comes to the end of the Career Award, she has obtained other sources of funding to continue her laboratory’s research, which focuses on understanding the mechanisms that regulate the function of hematopoietic (blood-forming) and myogenic (muscle-forming) stem cells so that their potential can be exploited for treating diseases such as cancer, anemia, muscular dystrophy, and diabetes.
Amy and her group have already made several important contributions to knowledge regarding these tissue-specific stem cells. Most recently, they identified a gene responsible for the division and movement of hematopoietic stem cells (HSCs). The ability to control the expression of this transcription factor, EGR-1, could lead to significantly improved success rates in bone marrow transplants, which are already the most common therapeutic use of stem cells.
Science writer Ernie Hood spoke with Dr. Wagers shortly after her April 10, 2008, Cell Stem Cell publication about EGR-1.
Amy, tell us about your background—where you grew up, and how you developed an interest in science…
I grew up all over the place. I was born in Ohio, but I lived in a couple of places in Ohio, North Carolina, Georgia, California, Arizona, Maryland, Kansas, Illinois…by the ninth grade, I was in my ninth school. But I knew by the time I was 10 years old that I wanted to be a scientist, although I didn’t decide to be a stem cell biologist until I was in graduate school.
Were you an Army brat?
No, my dad was an electrical engineer, and now he actually works in a real estate office, so I guess that moving around did something.
How did you settle on stem cell biology as the focus of your research?
I was a graduate student in immunology. I chose immunology in large part because I had a fantastic undergraduate immunology instructor [at Northwestern University] named Sue Pearson. She really inspired me to go into immunology, but while I was doing my graduate work in immunology, kind of finishing up and thinking about where I was going to do postdocs, I got a call from my mom. She said, normally I wouldn’t forward this kind of person, but it seemed kind of important. It was a call from the bone marrow transplant registry telling me that I had matched for a transplant with a patient, who remains anonymous. We went through the process of continuing to make sure that there was a real match. In the end, this person decided not to opt for the transplant, but as a result of that I actually did a lot of reading about bone marrow transplants and stem cells, and got really interested and excited about it. I decided that was where I wanted to do my postdoc work.
So I worked first with Irv Weissman, one of the leaders in stem cell biology and hematopoietic stem cells, particularly in bone marrow transplants. That’s how I got started in the field.
So the transplant itself never actually happened?
No, but every time I move, I update my information in the registry just in case that person is still out there…you never know.
Do you think the therapeutic use of stem cells will ever live up to the hype we’ve experienced in recent years? Is the potential as enormous as we’ve been led to believe?
I think the potential is enormous, but it is a concern whether the time line will be what has become the hype around it. As you know, all of these things take time. Obviously there is a lot of attention being focused on stem cell biology that really puts the pressure on to deliver real therapeutics in a very short period.
The range of time that it took to develop bone marrow transplants was more than 20 years, so you can expect at least as long a time line for other applications of stem cells for transplantation.
Applying stem cells as tools for drug discovery may be a nearer-term application, and I think there is a lot of promise in that arena as well, because they provide a model system for a disease or a model system for normal cell development that one can interrogate in a culture dish. So that is a much higher-throughput mechanism for looking for new pathways that might modulate disease or function of cells.
Tell us a bit about the stem cells you work on…
I work both on hematopoietic stem cells and skeletal muscle stem cells, which are a distinct population that lives in the muscle and is responsible for muscle growth and regeneration.
We focus primarily on what I would call tissue-specific stem cells, to contrast those from embryonic stem cells (ES), or the newly described induced pluripotent stem cells that Shinya Yamanaka [a professor at Kyoto University in Japan] developed the technology to generate with transduction of cells with four factors that convert them to a pluripotent state. In those cases, in the ES cells and the pluripotent cells, they have the potential to generate multiple different tissue types, probably all of the types in the body. The tissue-specific cells that we work with are committed to a particular subset of those types of cells.
And in the case of hematopoietic stem cells, they’re blood cell-generating?
They’re blood cell-generating, right, and the muscle cells are muscle cell-generating.
HSCs are already the only stem cells actually in widespread therapeutic use today, correct?
They’re the only cells that have been used in a purified form in a clinical setting. In skin grafting, you’re taking advantage of resident epidermal stem cells in those grafts. That would be akin to a bone marrow transplant, where there are stem cells within the graft, which also contains other types of cells.
What are the main obstacles to even more effective use of HSCs as therapeutic agents?
One of the major hurdles is getting enough cells. This can be a problem for autologous transplantation, where you’re taking the patient’s own cells after mobilization, then removing them, treating with chemotherapy, and then reintroducing those cells. Some people fail to mobilize effectively, meaning when you treat them to peripheralize the stem cells for collection from the blood, you don’t get enough cells, so they can’t go on to transplant.
Having enough cells to collect is also an issue of having the right kind of cells, so in cases where one can’t do autologous transplantation, you need an unrelated donor, and finding a match is often a limiting factor. And then getting the cells to engraft quickly so that you can avoid periods of immune deficiency that make you susceptible to infection is also an issue.
With all of that in mind, tell us about the discovery announced in your article in Cell Stem Cell…
As we were just discussing, two of the issues around transplantation are having enough cells and getting them to engraft quickly, and being able to collect enough cells. The exciting thing about this transcription factor that we described, and its phenotype in HSCs, is that it seems to control expression of genes that regulate the numbers of cells in the bone marrow, and also their movement into the peripheral blood, and their proliferative rate.
You could think about transiently targeting this factor or the genes that it regulates in order to enhance peripheralization of stem cells for collection, or to speed engraftment by driving stem cell proliferation in the early phases after transplantation.
So the discovery of this transcription factor could potentially lead to improvements in the effectiveness of this type of transplant?
I take it you used knockout mice lacking EGR-1 to give you proof of concept?
We did, we used knockout mice.
How did you discover what you believed to be the controlling role of EGR-1 in the first place? What was the scientific process that led you to this particular discovery?
We had been looking for genes that were differentially expressed in different physiologic states of HSCs. We particularly compared the normal, resting stem cell that we purified from the bone marrow to a stem cell that we isolated from mice that had been treated with drugs that caused the cells to proliferate and to migrate out into the periphery.
The idea was, we timed this so these drugs, when you deliver them, cause the cells to go through a very reproducible, stepwise progression, where you know at a certain time point after drug treatment they’ll be at a certain stage. So we timed this to isolate cells at just the stage where the stem cells were expanding very rapidly and just about to leave the bone marrow and enter the circulation. And so the prediction is that by comparing the expression of genes in these rapidly dividing, just premigratory stem cells versus a population of cells that was resting and resident in the bone marrow, we could find genes that were involved in stem cell proliferation and migration.
In that comparison, we hit on EGR-1, which is the transcription factor that the paper deals with, and found that it was dramatically down-regulated. We see the expression dramatically reduced when you cause cells to divide and migrate. That’s what led us to look at the knockout mice to see whether, if we looked specifically at the stem cell population in those animals, it would also show this phenotype of enhanced division and enhanced peripheralization of HSCs. And it did.
It sounds like once you honed in on it, it looked like a smoking gun. How did you identify that EGR-1 was the gene at work? Did you do whole genome association studies?
Yes. We were comparing differences in gene expression by quantitative PCR. Several of the genes in this family have been implicated in regulating HSC function. But this particular one hadn’t been directly implicated in HSCs. Deletion of some of the other family members will actually lead to leukemia in mice.
But this one…
This one doesn’t seem to. There was another study published recently in the lab that suggests that haploid sufficiency, or losing one copy of this gene, can, with collaborating mutagens, promote leukemogenesis. But we’ve never seen any spontaneous tumors arising in our mice.
It’s a nice thing because if you wanted to think about targeting this gene for therapy, obviously you would be discouraged from doing that if the sufficiency led to leukemia. So from this we know that even long-term loss of function of this gene doesn’t cause tumor development, so transient targeting of it is a reasonable approach to consider.
Isn’t it unusual for a single gene to have such a profound controlling impact on a particular function? Or is this sort of a master regulator gene within a whole network comprising the pathway?
The short answer is, we don’t know yet, in the sense that we’re just starting to study how this gene interacts with other genes known to regulate HSCs. But my suspicion is that it’s a master regulator controlling these processes. In ongoing studies we’ve begun to identify the subset of genes that we think it regulates, and those are looking very interesting as well.
One of the things we haven’t talked about yet that I think is particularly interesting with this gene is that because it is a transcription factor, it has the potential to control multiple other factors. So it probably does act as a nodal point for control. And it’s the only transcription factor yet identified that regulates the localization of stem cells in the body. There are other genes that have been reported to control the migration of stem cells. Those include receptors on the cell surface and signaling molecules in the cytoplasm. But this is the first transcription factor, and the nice thing about that is that it really does get you to what might be a gene network controlling those processes.
It seems to have a specific, unique role in this sense, because it acts to coordinate that migration with the division of the cells, with the replication of the cells, and that’s unique because other factors that when deleted perturb the proliferation of HSCs don’t lead to this change in their localization. So it looks like this factor is commanding two programs that are independent but coordinated through the activity of the transcription factor. That’s an interesting thing to think about in the context of why HSCs might want to coordinate their migration with their proliferation.
Now that you’ve been able to identify this particular gene and its seeming master regulatory role, what’s next?
We’re going on to try to figure out the interacting partners as well as the targets of the gene, because we think that will give us more refined targets for manipulating stem cell function in the context of mobilization. We’re also looking at how this factor might be playing a role in the ways that the stem cells interact with other cells in their environment, whether it’s changing fundamentally the progeny cells or the nonhematopoietic stromal cells that interact with these.
What is the therapeutic potential here? Is that something you will be pursuing actively?
Yes. What we’ve done is identify that in a mouse model, where there is continued inactivation of this gene, we have this effect on stem cells. The question is, if you perturb the gene’s function transiently, would you get the equivalent effect, which is what you would want to do therapeutically. You obviously wouldn’t know that you wanted to inhibit this gene all the time.
Might you use RNA interference, or gene silencing?
Right, exactly. We’re going to try this directly by RNA interference. In the long term, I’m not sure that RNAI will be an effective therapeutic, but you could think about developing small molecule antagonists against this factor, which is a zinc finger protein. There have been some successes with developing small molecule antagonists against zinc finger proteins.
The other route to go, which is complemented by what we’re doing now, is to figure out what are the targets of the transcription factor that really matter for these behaviors that we care about. Those may be more amenable to targeting by drugs. So figuring those out may lead us to the particular therapeutics that we’d want to employ.
With that in mind, as you’ve discussed, EGR-1 is apparently responsible for a variety of other functions as well as the ones we’ve been talking about…with the potential for therapeutically affecting it, whether it’s by RNAI or small molecule antagonists or whatever, how difficult do you think it would be to regulate its expression without causing potentially dangerous unwanted effects? Or is that another reason to look at some of the downstream targets?
I think the fact that we have knockout mice that lack this transcription factor entirely and that seem to behave perfectly well—they don’t have any shortened life span, they don’t have any blood malignancies—argues that we will be able to down-regulate its function without any deleterious effects on the blood system or on other organ systems.
These animals have a mild thymic defect, a mild defect in the production of T lymphocytes, that doesn’t seem to impair the production of mature cells, and they have female-specific sterility that has to do with the production of luteinizing hormone. That’s reversible by providing luteinizing hormone, and presumably in the therapeutic setting it would be a transient thing.
How quickly do you see this potentially proliferating into human applications? Is it something that’s going to need quite a bit more basic research in mouse experiments, or do you think we might actually be able to put this to therapeutic use in humans fairly soon?
I think it depends on whether we can identify a small molecule antagonist to this factor right away. We’ve definitely been in contact with a few groups that are looking into small molecule inhibitors of zinc finger proteins, of which this is one, to see if we might be able to utilize those.
I think the outcome of those experiments will really let us know whether this is going to be a near-term sort of approach, or whether we’re going to have to go back to the basic biology of discovering additional targets and finding the inhibitors of those targets, which would obviously make it a longer-term approach. Obviously, it also will depend on the toxicity analysis of any small molecule inhibitor we were able to discover that worked against this protein. So it’s hard to predict right now.
So you’ve identified a pretty attractive target, and now you’ve just got to find a drug that works, right?
This may also have implications for treatment of type 1 diabetes…how would that work, in theory?
Because type 1 diabetes is a disease that’s mediated by immune attack on pancreatic beta cells, one of the primary issues with type 1 diabetes is getting the immune system to stop the assault on the pancreas. In mouse models, at least, it’s been shown that one can stop this by bone marrow transplantation.
The problem is that, first of all, in human patients, by the time you recognize that there’s a need, most of the beta cells are destroyed, so they would still need to be grown back from a source of replacement cells. One could accomplish that with islet transplantation, although there are limitations to the number of cells you can get there.
The other issue is that under the current approaches of bone marrow transplantation, the process is far too dangerous and potentially toxic to justify subjecting a child with type 1 diabetes to that kind of transplantation protocol. But if one could make transplant less toxic, more tolerable, and less potentially dangerous, then one might consider performing that kind of procedure on a type 1 diabetic. So in every way that we can improve transplantation, we could bring it closer to being useful for treatment of diabetes in that way.
Where is your research headed from here?
We’re finding targets for this transcription factor, and we’re also looking at whether transient inhibition of the transcription factor has equivalent effects on the stem cells as this constant deletion of the gene has.
Amy, you are still at an early stage in your career? What has been the importance of having the Burroughs Wellcome Fund Career Award, and the subsequent awards you’ve received? In these days of flat funding, you’re very fortunate.
Yes, it’s been absolutely essential. We would not have been able to do these things without that funding. My lab is still unfunded by the NIH, so we are surviving based on our success with raising funds from non-NIH sources.
That’s amazing—no funding from the National Institutes of Health at all?
Well, I haven’t had an RO1. I do have a pilot feasibility grant that’s funded through a Center grant to the Joslin Diabetes Center. That amounts to $35,000 a year for two years. That’s been my NIH funding.
So it’s these foundation grants that have really enabled you to do this important work?
Do you see that situation continuing?
I certainly am working very hard to get funding from the NIH. I actually have three grants now pending with them. It’s a difficult climate, particularly for new investigators. At the same time you’re trying to establish your research, you’re being held to standards and comparisons with senior investigators who have had their laboratories for a very long time. As much as there is every attempt on the part of the review committees to take into account people who are new investigators, the problem is there just isn’t enough funding to go around. Everyone is in an incredibly difficult position at this point.
According to a new report about NIH funding levels, the rate of success for funding for first-time RO1 submissions is now 12 percent, down from 29 percent in 1999. So it’s pretty serious.
It certainly is, and it seems to be fortunate for people at your career stage, making that transition into independence, that these types of foundation monies are out there…
Yes. They’ve always been incredibly important, but particularly now they’re essential for supporting new people—particularly people who do science in an interdisciplinary fashion or in new areas. That makes it even more difficult to win funding from the NIH, particularly when you’re a new investigator without an established track record. If you propose discovering something novel, it raises concerns.
I would hope that the success you’ve had would start speaking for itself in those terms.
I hope so, too!
Amy, for someone your age, just 34, you’re doing some phenomenal work. How are you managing to maintain a balanced life in the course of this? I saw that one of your hobbies is actually the flying trapeze, of all things…
Yes, but sadly I haven’t been to the trapeze school in almost a year. I’ve got to get back there! My lab gave me a couple of hours at the trapeze for my Christmas holiday party gift.
It’s tough. It takes a great deal of effort to keep things running, but part of it is enjoying what you do, so that it doesn’t seem like such a drag working weekends.
You’re not going to be running away and joining the circus on us, are you?
No, not yet!
Setting the record straight
The Scientist (Jan. 1, 2008)
She's driven to succeed in stem-cell work
Boston Globe (May 3, 2005)
Ernie Hood is a freelance science writer and editor based in Hillsborough, NC. Mr. Hood hosts a weekly science radio program, Radio In Vivo: Your Link to the Triangle Science Community, on WCOM-FM in Carrboro, NC. With more than 80 hours of programming now archived at its website, Radio In Vivo serves as an information resource to the public about the important, exciting, and innovative scientific work taking place in the Research Triangle area of North Carolina, an internationally-recognized focal point of research and development in all scientific arenas.