PENNINGTON BIOMEDICAL RESEARCH CENTER

Bring up the word "stem cells" in conversation, and you are likely to get a variety of reactions, from "What's that?" to "Aren't they going to cure all kinds of diseases?" to "Isn't that what people want to ban?"

The use of stem cells in medicine and the field of research surrounding them is still so new that it has become regular subject matter only among interested parties: physicians, researchers, and those, such as politicians, who are struggling with the ethical issues of stem cell use. The world of stem cell research is still one big question mark for most of us.

Three investigators at the Pennington Biomedical Research Center, a campus of the LSU System, are hard at work seeking answers to the questions about stem cells and what their ultimate place may be in the world of health and medicine. But interestingly, if you bring up the word "stem cells" to them, they may also ask, "What do you mean?"

Although it's become common to hear media reports about scientists prodding stem cells to "turn into" other tissue, like bone, cartilage, nerve cells, skin and the like, scientists are still honestly debating among themselves about what is or is not a "stem cell," where they originate, and how they know if they have one or not.

Pennington researcher Jeff Gimble, who holds both an M.D. and a Ph.D., made international news recently when stem cells he harvested from adult human fat-with a little prodding from him-turned themselves into human bone. He separated the stem cells from regular fat, grew them in the lab until he was satisfied they were healthy, then placed them on a small chip of bone which he then slipped under the skin of a mouse. The stem cells took their cues from the bone chip and from the normal chemical messages they received from a living organism and did what they were "told" to do ... they turned to bone and started growing.

Dr. Ken Eilertsen, on the other hand, is scrutinizing stem cells that form well before adulthood; his cells have the potential to form an embryo and, under the right conditions, subsequently produce a living organism. These are referred to as totipotent stem cells, and there the story gets a bit more complicated.

Under normal circumstances, when a sperm fertilizes an egg, that single cell is totipotent, meaning it has the potential to form the total range of cells and, ultimately, can grow into a living organism-the mother lode in the world of stem cells.

Thus every animal starts off, quite literally, as a single, totipotent stem cell. However, that ability to form all cells-called totipotency-fades as cells within the embryo begin to differentiate into layers. Totipotential gives way to a lesser ability called pluripotential. Pluripotent cells no longer have the capacity to develop into an organism. Rather, they will form the different cell types needed within the whole organism. For example, pluripotent stem cells that reside in a place in the embryo called the inner cell mass can differentiate into three distinct layers: the endoderm, ectoderm and mesoderm, meaning simply the inner layer, outer layer and middle layer, respectively.

Cells in each layer become restricted to forming specific types of tissue. For example, the endoderm yields the liver, pancreas and other internal organs; the ectoderm becomes the nervous system; and the mesoderm matures to muscle, bone and cartilage.

Eilertsen spends his time trying to clearly understand and document the specific genetic and molecular processes that create totipotency and that are subsequently turned off or suppressed as the embryo matures. If he learns what turns totipotency off, he may find the key to turning it back on as needed.

The question driving most stem cell research now is, can scientists learn to harvest stem cells from whatever the source and coax them into becoming the tissue they want?

Gimble's conversion of adult, fat stem cells into bone offers one possibility. One day those cells may lead to a source of bone tissue to repair crushing injuries where not enough bone is available to graft. The injured person could donate his or her own fat stem cells, thus avoiding the tissue rejection associated with transplants. Similarly promising results are becoming more and more common in the scientific literature, which now seems to carry a steady stream of news in which researchers announce their latest results in grooming stem cells to turn into bone, cartilage, muscle and other tissue.

But Gimble's cells are only multipotent; his fat stem cells may never become anything other than fat, cartilage and bone. Not a problem, really, just a fact.

"You don't have to have a totipotent cell to repair a lot of things," Gimble notes. "You can repair many types of tissue with one type of stem cell."

Blood marrow is an example of how one source of stem cell can form multiple tissues. Bone marrow contains multipotent cells called hematopoetic cells-cells which form blood, primarily. Gimble and others can mark these marrow cells with special dye, however, and find them later in the brain, nervous system, liver and muscle.

Thus the difficulty of the questions: Exactly what is a "stem cell"?, and, How do you know when you have one? It is not as simple as peering through a microscope and identifying one. And, to make the matter more intriguing, Gimble has seen evidence that his multipotent fat stem cells might actually be more like pluripotent cells-able to convert to a wider range of tissues. Bone marrow also contains pluripotent cells, a complicating factor in sorting out the stem cells there. And other layers of complexity are leading researchers into a wide field of cells, each with some potential to convert to specific tissue, but hard to define and describe in an exact manner-a manner that will allow all scientists to know they have isolated the exact type of stem cells they need for laboratory or clinical use.

Eilertsen likens the search for stem cells to throwing a handful of similar looking seeds into a garden. You may not know which are the red flower seeds (representing stem cells) until after the flowers bloom. In both Eilertsen's and Gimble's work, the scientists may be isolating and manipulating stem cells and non-stem cells alike, knowing what they started with only after a tissue conversion takes place.

Both scientists are trying to understand the range of cells and develop the means to positively identify them. And, more subtly, to identify the original stem cells used to grow new tissue, so that-like continually saving a ball of starter dough to leaven the next loaf of bread-a researcher might harvest the exact same kind of cells a second time, or third, and coax them into a different tissue type.

The discussion of the exact definition of a "stem cell" and the quest to understand each and every type will continue in scientific circles for years. In the mean time, few doubt the use and benefits of the cells now dubbed "stem cells." One of the major medical uses is to repair diseased or damaged tissue using stem cells donated by the patient. The cells would be guided to convert to the needed tissue type and implanted in the patient. That process holds the promise that the patient's body would not reject the new tissue as often happens in traditional organ transplants, so a graft or implant may be much more effective.

Reverse rejection may also be eliminated. Take the case of kidney damage. A patient in need of a new kidney faces the fact that the new kidney will have kidney cells and immune cells. The kidney may therefore try to fight off the patient's body at the same time as the patient's immune system tries to fight the new kidney. If doctors could find the patient's own stem cells that have the potential to form kidney cells, technicians could convert the stem cells to kidney and allow the patient to receive his or her own tissue back in another form. That would eliminate the problem of organ rejection.

Scarring of skin and other tissue may also become a problem of the past. A third stem cell researcher at Pennington is investigating the effects of stem cells on healing. Dr. Barbara Gawronska-Kozak began her stem cell research after making a peculiar and unexpected discovery during a procedure used to mark laboratory mice. Technicians create tiny holes in the ears of mice similar to those many people use for wearing earrings. These marker piercings are normally made in the ears of mice at one month of age and are permanent. However, Kozak noticed in one particular strain of mice-a strain with a deficient immune system-that the holes would completely heal over, with no scarring.

Since that complete healing required creation of cartilage, fat, skin and new blood vessels, it appeared as if functional tissue was being completely renewed in the ears of these unique adult mice, a process called regeneration. Yet, complete regeneration normally occurs only in animals such as amphibians, not in higher vertebrates.

Kozak's experiments to understand the cellular basis of this tissue regeneration has led her to isolate adult stem cells from the ears of mice that, like the stem cells found in Dr. Gimble's fat tissue, are able to differentiate in tissue culture into fat, cartilage, bone, and even "myocytes," the heart muscle cells that actually beat. Now, Kozak wants to know how mice with defective immune systems are able to regenerate tissues. Most strains of mice possess stem cells that are able to make the cell types necessary for tissue regeneration, but for unknown reasons only the environment present in immune deficient mice permits the emergence of new tissues when the ear is wounded.

It is a similar question that informs the work of both Eilertsen and Gimble: what are the molecular and genetic reactions that tend to create and control stem cells? Eilertsen's hope is that if he can learn exactly how to identify totipotent stem cells and document exactly which molecules, which genes or which proteins determine totipotency, then researchers may be able to develop stem cell-based therapies with a high probability of success. Also, the knowledge could improve a procedure called "somatic cell transfer" or SCNT. (The famous "cloning" technique used to reproduce livestock.)

In SCNT, Eilertsen can take a cell that has already become a specific tissue type and restore its totipotency. He transfers this new stem cell into an egg that has had its own DNA removed, and the egg then matures into an organism guided by the implanted DNA. This is the basic method to reproduce an animal with a known genetic make-up-an animal that might be programmed to produce therapeutic proteins in its milk, or supply organs for transplantation in humans, for example .

Currently, however, scientists face more failure than success in guiding SCNT cells to become actual animals or stem cells to become specific tissue types (or to become whole animals), partly because they don't know all the specific molecular and genetic steps in the process of stem cell growth and differentiation. Eilertsen is studying the reprogramming of cells at the molecular level, so that scientists may learn how to increase the likelihood of successful stem cell conversion.

When Eilertsen takes DNA from an adult pig and places it in a pig egg, what does he really have? Is the new cell like a normal fertilized egg, needing only the environment of the womb to successfully multiply and mature? Or is the new cell trying to behave like an adult but trapped in a new embryo? Developmental biologists have typically treated these SCNT fertilized cells as normal embryos, and attempted to duplicate the environment of the womb in hopes they would grow and mature.

Eilertsen, on the other hand, believes the newly fertilized cells may behave more like the adult, DNA-donor cell. He has studied thousands of genes in embryos produced by SCNT, which typically do not develop into animals very well. He has identified more than 100 genes that are not as active in his SCNT-generated pig embryos as in normal embryos, and these genes are involved in several functions, indicating that true totipotentiality is modulated by a whole group of genes and the proteins they ultimately produce.

Further, although the activity level of the genes in SCNT-generated embryos is unlike a normal embryo, the activity is very similar to its adult donor cells. This means, perhaps, to increase the survivability of cloned embryos, technicians may need to regard them more as adult-like cells with unique needs and create a custom lab environment, not merely duplicate the environment of the womb.

Gimble, working nearly alongside Eilertsen, is trying to expand what we know about stem cells that were once believed to be restricted to producing just a few tissue types. One reason is that many people are deeply concerned about the use of human embryos as a source of stem cells. If Gimble and others can discover more adult stem cells and guide them to more purposes, much of that concern will be bypassed.

There are still more questions than answers, even when it comes to determining what exactly is a stem cell. But the enormous potential of stem cells, Gimble notes, means it would be unfortunate if any existing avenues of research were prematurely closed.