Tapping into Blood-forming Stem Cells
The power to fight aging, knock out cancer, and regenerate tissue may have been hiding inside our bodies all along.
Scientists knew primitive powerhouses lived in our blood and bone marrow, but recent studies have uncovered them in organs never before thought to house themthe brain, heart and pancreas, to name just a few.
What are they? Stem cells: the self-renewing building blocks of the human body.
In his 10 years at the University of Kentucky, Gary Van Zant has probed the bone marrow's stem cells, and his findings have implications ranging from the immediate (better cancer treatment) to the seemingly sci-fi (organ regeneration). But what makes Van Zant's research unique among scientists investigating stem cells is his angle: aging.
He's looking at four main questions: What is the link between cancer and stem-cell aging, and what impact does that have on bone marrow transplantation? Does age play a role in a stem cell's ability to find its way home to the bone marrow after transplant? And what genes are involved in regulating stem-cell population size?
On Van Zant
Stem cell researcher Michael Clarke, a University of Michigan colleague of Van Zant in the mid-'90s, sums up Van Zant's research: "Gary has pioneered our understanding of the role that stem cells play in aging. He was the first to show that aging of an organ, specifically the bone marrow, was a consequence of loss of stem-cell function. He then designed elegant genetic studies to identify specific genes that modulate stem-cell aging."
But before this "elegant" genetic research, in which Van Zant inserted human stem cells into genetically modified mice, his focus was blood. "I've always been interested in stem cells because they generate all of the cells in the circulating blood, and they have to do so for our entire lifespan because mature blood cells have very short lives," says Van Zant, who holds a Ph.D. in cell biology and hematology from New York University.
Gary Van Zant has spent a decade at UK studying age-related changes in bone marrow stem cells. Van Zant is a professor of internal medicine and physiology, and serves as director of the stem cell processing laboratory for the Markey Cancer Center Bone Marrow Transplant Program.
"Red blood cells live about 120 days, platelets [cells that cause blood to clot] live seven to 10 days, and granulocytes [a type of white blood cell] live only a few hours. Stem cells need to constantly replenish cells that are lost through regular wear and tear.
"So the question I began pondering was 'Do stem cells themselves wear out?'"
Not only has this question fueled his basic research, but the answer has immediate practical application to the clinical part of his job as director of the Stem Cell Processing Laboratory for UK's Markey Cancer Center Bone Marrow Transplant Program. He explains: "We harvest and freeze human stem cells that are going to be transplanted back into patients in the course of cancer treatment. To improve this process, the question I needed to answer was, 'Should we be concerned about how old the stem cells are that we're harvesting and transplanting?'"
Stem Cell Isolation
Blood-forming stem cells, known as hematopoietic stem cells (HSCs), have an identity problem.
First, they're rare. Scientists estimate that only one in every 10,000 to 15,000 bone marrow cells is an HSC. They live in the bone marrowthe soft, sponge-like material found inside bonesbut they also circulate, at rates of one in 100,000 cells, in the bloodstream.
Second, you can't pick an HSC out of a line-up: they have no distinguishing features to separate them from look-alike blood and bone cells. Van Zant says, "This bedeviled hematopoietic researchers for a long, long time because HSCs are so rare, so nondescript, and have no markers of differentiation because they’re primitive. It took many years before science could make stem cell purification routine."
The breakthrough came with a new technology: "Flow cytometry has been around since the 1950s, but it really took off with the development of monoclonal antibodies in the '70s," says Van Zant. Flow cytometry takes advantage of cell markers, specialized proteins on the surface of each cellcalled receptorsthat have the capability to adhere to other molecules. Cells use these receptors and the molecules that bind to them as a way to communicate with other cells and to carry out their proper function in the body. Monoclonal antibodies were developed for all kinds of cell markers, which allowed scientists to identify and separate different cell types.
The antibodies were coupled to fluorochromeschemicals excited by different wavelengths of laser light. "In an extremely rapid fashion, flow cytometry interrogates large numbers of cells on a cell-by-cell basis using laser beams." Van Zant explains, "Any lab studying hematopoiesis worth their salt has access to a very good flow cytometry facility. And we do here.
"Stem cells have certain markers that no other cells have, and, in addition, they don't have markers that most other cells do, so you can use this sort of yin and yang approach called negative/positive selection to isolate them."
He pulls out a chart of results from the last blood sample he ran through the flow cytometer. "Here, in a two-dimensional chart format, we've analyzed 50,000 cells in 40 seconds. You can take your bone marrow or blood sample into the flow cytometry room and walk out with a pure population of stem cells. Then you can study them in isolation from all of the other cells, and that ability has greatly advanced our work."
For more stem cell basics read Stem Cells 101.
Jeff Yates, a fourth-year doctoral student, is working on a dissertation on how HSCs respond to stress and how this response is related to life expectancy. "Part of the admissions process for graduate students in the UK College of Medicine is faculty interviews. Dr. Van Zant was one of mine. He really sold me on the clinical significance and promise of his research. He's a pioneer in defining how aging alters the function of stem cells. And he's a big proponent of giving his students total freedom in designing and carrying out their research projects," says Yates.
Age = Cancer
The biggest risk factor in cancer development is age, Van Zant points out, "and I'm talking about all kinds of cancer. Between the ages of 40 and 80, there is an exponential increase in cancer incidence. After age 80, it levels off.
"In cancers of the hematopoietic system, namely leukemias, the most studied is acute myeloid leukemia, or AML. AML is three times more likely to occur in someone age 65 versus someone 35."
Where does leukemia come from? Stem cells. "Studies have indicated that the original cell in AML was a primitive stem cell that took a wrong turn on the developmental pathway. What happened?" he asks. "Most likely a buildup of mutations."
Van Zant's 10-person team, supported by a number of National Institutes of Health grants, is studying the age-related genetic changes that turn a normal cell into a cancerous one. "These genetic alterations can include independence in growth signaling, escape from apoptosis [cell death], and endless ability to replicate.
"A buildup of mutations takes place over the lifespan of an individual. And that buildup is due in part to repeated rounds of cell division that provide opportunities for editing errors in the DNA. That genetic damage is passed on to all of the stem cell's progeny. The inappropriate differentiation and proliferation of that damaged stem cell results in a tumor."
Van Zant says the failure of current cancer treatments just might be their inability to attack damaged stem cells. "Standard cancer treatments are aimed at end-stage cells, and they zero-in on them by exploiting the fact that end-stage cells divide rapidly." This rapid division causes the tumor to grow, but all the while the mother stem cell is relatively quiet. "She's not dividing much because she doesn't have to. Her progeny are doing the work.
"So it's a common theme: you treat the tumor with one of these drugs, and the tumor shrinks. It remains small for some time, but then it grows back. And a possible reason, at least in some cases, is that you haven't touched that 'quiescent' stem cell. It just takes that stem cell a little while to generate progeny again to grow the tumor."
The questions Van Zant is asking about stem cells apply not only to the link between cancer and aging.
"My over-arching philosophy is that the stem cells in various organs of the body could well be the focus for the age-related declines we see in those organs. Each organ suffers age-related decline in activity, but we haven't fully established what causes decline in a stem cell. Is it the ability to respond quickly to a signal that more mature cells need to be produced? Is it the inability of stem cells to divide in a rapid and appropriate way? We just don't know yet."
For more, read Bone Marrow Transplant 101.
"As a principal investigator, I have a tremendous responsibility to maintain funding for these projects because they support all of the people in my lab. I've got to look after my research family," says Gary Van Zant. Back row (left to right): Ying Liang, Adrienne Ellis, Jeff Yates, Barry Grimes, Carol Swiderski, and Amanda Waterstrat. Front row: Mindi Haley, Debbie Bell, Erin Oakley, Van Zant, and Alison Miller.
Stem Cells, Stem Cells Everywhere
The human body is filled with stem cells.
"It used to be thought that stem cells were found in only a very few organs of the bodythe bone marrow, the gut lining and the skin. This was the dogma for a long, long time," says Van Zant. "Recentlyand this is a huge area of research right nownumerous organs in the body are found to contain stem cells."
There are neural stem cells: "Of course the big push in that field is to find a way to repair central nervous system damage from accident or diseaseAlzheimer's, Parkinson's. There are a number of childhood diseases that affect the central nervous system, and if you could go into the stem cells and repair the damage, it would have tremendous implications."
The pancreas has stem cells: "Diabetes is a huge problem in this country. Imagine if you could go in and fix the insulin-producing cells with stem cells."
Heart muscle contains stem cells: "It was thought that if you had an infarctiona great big chunk of your muscle was damagedyou were pretty much out of luck. Now it's becoming apparent that quite a bit of regeneration occurs in the heart, and cardiac muscle stem cells apparently play a role in some degree of turnover."
Another exciting, albeit controversial, area of stem cell organ research is transdifferentiation, which Van Zant defines as "the ability of an adult stem cell to switch specificities to suit the environment in which it finds itself."
He sets up a transplant scenario: "Let's say you get a bone marrow transplant from a sibling. In half the cases that graft is going to be sex mismatchedbecause it's just as likely that your brother would be matched to you as your sister. So you, as a female, get a bone marrow transplant from your brother.
"In a real case, a woman got a bone marrow transplant from a man. Years after the transplant and after that woman died, the doctor performing the autopsy wondered if the transplant had replenished any tissues other than the bone marrow. And low and behold, he looked in her heart and there were cardiac cells that were male. They could have only gotten there from the bone marrow cells that were transplanted into that woman. This is one of the strongest arguments for transdifferentiation."
Van Zant adds there's also evidence that scientists can take an adult stem cell from bone marrow, transplant it into a damaged liver, and replenish the liver cells. "There's also evidence that bone marrow stem cells can regenerate, or at least generate progeny, in the brain.
"But why doesn't this happen normally? Why don't the stem cells floating around in your blood 'get off the train' and migrate into damaged tissues? Well, there are obviously things we don't understand, but the data's pretty tantalizing that, in fact, under some conditions, stem cells from the bone marrow can fix damage in other organs."
The young stem cell population (a) starts out with a big reserve of resting (quiescent) cells, a few dying (apoptotic) cells and a healthy population of active cells that produce more of themselves and enough white blood cells (the lymphocytes and granulocytes) to protect us from infections. In (b) and (c), aging and a stressful condition that requires extra blood cells conspire to severely damage the ability of stem cells to do their job. Aging and stress cause more to die from accumulated cellular damage, and the survivors produce fewer lymphocytes.
Mice as a Model
"If we measured the number of HSCs in the bone marrow of two 'normal' humans, like you and me, we may find that the difference is about 100-fold between us. And yet, we’re both apparently normal. How can that be?" Van Zant asks, more animated with each word. "And what are the ramifications for the chances of one or the other of us getting leukemia or aging faster? These are the kinds of questions we hope to answer with mice.
"Mice mimic the vast majority of biological features that we find in humans, including aging. Only it's on a much accelerated chronological scalein two years a mouse goes through a series of processes that are mimicked in humans over eight or nine decades," he explains.
"In the bone marrow itself, the age-related changes in HSCs are very similar. In humans the bone marrow becomes hypocellular, that is, the number of cells in a given volume of bone marrow decreases with age. The same is true in the mouse. The nice thing about mice is you can get a group of mice that are genetically identical."
One such group, or "strain," of mice is immunodeficient (labeled by scientists as NOD/SCIDnonobese diabetic/severe combined immunodeficient). "This is a strain in which the immune system is completely wiped out. They have no immune response to anything. Scientists have used that mouse as a transplant model into which they've put human cells, and that's what we've done.
"You can give a mouse virtually the same treatments that are administered to a human in cancer therapy, and you can do a bone marrow transplant in a mouse that's virtually identical to one in a human. And the recovery patterns are very similar."
These similarities have allowed Van Zant's team to make two important discoveries.
"We've taken bone marrow stem cells from different age groups: people age 80 and older, middle age, adolescent, and newbornsfrom umbilical blood. We can study the differences in these stem cells very precisely by putting these cells into NOD/SCID mice. We can get actual numbers of stem cells, and see how quickly they repopulate the bone marrow.
"We've learned two interesting things. First, old bone marrow doesn't repopulate the NOD/SCID mouse very wellnot nearly as well as umbilical cord blood. And second, we have very convincing evidence that old stem cells have a greatly diminished capacity to find their way home to the bone marrow."
Scientists don't know a lot about stem cell "homing," and these are the questions Van Zant is tackling. "Is homing a random process? Is it just a scatter-gun approachthey go in, some of them hit the target, but 99.9 percent don't? Or can it be directed? Why do old stem cells find home less effectively than young stem cells?"
Van Zant says his findings so far are clinically important because they suggest that you may not want to bother harvesting stem cells from older donors. "Even if the stem cells themselves are 'good,' their age seems to automatically lower their chances of making it into the bone marrow."
Amanda Waterstrat, who earned a bachelor's degree from Lindsay Wilson College in 2003, says, "A strong interest in aging research led me to this lab, and I think I've found a wonderful place to begin my scientific career." And Erin James Oakley, a Centre College graduate from Cadiz, Kentucky, agrees: "Working in Dr. Van Zant's lab has been great. As a student, I've been exposed to a strong supportive network encompassing all levels of expertise."
The very method of Van Zant's research means he can't predict where it's going to lead. He explains: "We know these mice are different in a very reproducible way, but what possible genes can cause that difference?"
The first step to identify genes is zeroing-in on the appropriate chromosomes. (Humans have approximately 30,000 genes on 23 pair of chromosomesone of each pair you got from your mother, the other from your father. Mice have roughly the same number of genes on 20 pair of chromosomes.) "When you're looking at all possible genes, you never know where you're going to end up. And that's exciting."
The most recent source of excitement is a genetic discovery by fifth-year graduate student Ying Liang, who works in Van Zant's lab. Van Zant cautions that he can't divulge too many details about this discovery because he and Liang are pursuing a patent on it, but he comments, "Her work makes us almost completely certain that this gene, that had never before been shown to have an effect on hematopoietic stem cells, appears to be very important."
Fifth-year graduate student Ying Liang and Gary Van Zant are pursuing a patent on a gene that she discovered which plays a key role in expanding stem cell numbers.
Liang, a native of mainland China, says her work focuses on the regulation of stem cell numbers. "HSCs are very important in bone marrow transplantation. However, a big obstacle for this procedure [see sidebar on umbilical cord blood] is that inadequate numbers of stem cells are available for transplantation. Right now, we are looking for genes that might be responsible for expanding stem cell numbers, and we've already found a gene that may play such a role. I'm testing the functions of this gene by overexpressing it [purposely producing excess] in HSCs to see whether or not the stem cell population increases."
Van Zant's team has located another target gene on Chromosome 11. "We have a very strong candidate, Rad50, a gene vital to DNA damage repair. This is important because the long-standing damage-response theory argues that aging is the result of accumulated damage to the macromolecules of cellsprotein, lipids, nucleic acidsand that accumulated damage accounts for the physical manifestations we associate with age," he says.
Because stem cells are capable of spawning so many progeny, they must have "robust mechanisms of DNA repair. Defects in a critical DNA repair component such as Rad50 could seriously affect the ability of stem cells to functionparticularly in old age," Van Zant says.
"If stem cells can't supply the appropriate numbers of progeny, aging is accelerated. And if DNA damage is not properly repaired by Rad50, mutations may result in tumor formation."
It all comes down to this: "The natural course of aging and its undeniable association with cancer is not lost in a stem cell, despite its amazing ability to self-renew and restore tissue in times of need. Stem cells have tremendous potential. And we're here to help answer the basic questions that will lead to a new generation of cellular therapies."
"My first impression of Dr. Van Zant was that of a caring mentor, interested in the education and well-being of his students," says Alison Miller, a Centre College graduate student from Knoxville, Tennessee. "He has proved to be just that. Working in this lab has allowed me to interact with collaborators at other institutions, as well as travel to several international conferences. These interactions have helped me to shape my future career plans and to gain insight into other scientific problems in the field of aging."