Cell Isolation: E. J. Read (left), chief of the CC Cell Processing Section, watches as medical technologists Quyen Chau and Tamara Felton run the CliniMACS, a closed, sterile system that uses magnetically labeled antibody to separate enriched hematopoietic progenitor cells from a donor’s mobilized peripheral white blood cells. A typical donor sample, containing 40 billion white blood cells, provides about 400 million progenitor cells for one allogeneic transplant.

Radical new therapies using today’s government-approved embryo stem-cell lines may still be five or more years from trials in patients. In the meantime, NIH scientists developing therapies based on adult cells are making interesting strides in basic and clinical studies.

At an NIH workshop in late May, these investigators said they hope their real-world experience turning scientific concepts about stem cells into human cell-therapy trials will also ultimately facilitate embryo stem-cell treatments.

Speaking at the May 24 workshop, "Clinical Applications of Stem Cells at the NIH," John Barrett of NHLBI acknowledged that he had "a slightly crusading purpose" behind his talk—not just to show promising clinical trial results, but also to highlight the challenges of his 10-year effort to take a promising idea from theory to clinical trial.

Effecting Smooth Transitions

From this experience, Barrett sees a need for a core facility to assist what he prefers to call "transitional" research—the work needed to go from "proof of principle" experiments with human cells to the manufacture of an FDA-approved clinical-grade cell product.

A transitional research laboratory would facilitate interactions with industry partners to study and perfect the systems for cell separation, selection, culture, and preservation so that the techniques for making new cell products could be brought within reach of cell-processing laboratories and blood banks nationwide.

The Cell Processing Section in the CC’s Transfusion Medicine Department, which operates under rigorous GMP (good manufacturing practice) conditions, would be an essential partner in that effort, he emphasized.

Creating a transitional research facility might be expensive, he said, but the ultimate savings from transplants using sophisticated cell products that could emanate from such a facility would be enormous.

Barrett cited comparative costs within his own area of hematopoietic stem-cell transplantation for malignant diseases: The creation of complication-free transplants—purified stem cells free of cells that cause graft-vs-host (GVH) disease and enriched for immune cells that specifically react against troublesome viruses and the patient’s cancer—could cost as much as $20,000 per patient. But that expense pales in comparison to the possible $250,000 price tag for the prolonged hospitalization that often attends allogeneic stem cell transplantation and the cost of treating recurring disease if the transplant fails.

Cancer and Graft-vs-Host Disease

Barrett, along with NCI’s Michael Bishop, described research that uses hematopoietic stem cells in treating cancer patients.

Barrett uses allogeneic transplants of peripherally harvested bone marrow cells to treat leukemia. The HLA-matched donor stem cells repopulate the leukemia patient’s blood-producing system after their own cancerous blood cells are killed with anticancer chemotherapy.

But beyond that, Barrett’s team is trying to improve their patients’ odds by delicate application of one edge of a dangerous two-edged sword.

Investigators have seen that immune reactions by grafted donor immune cells can have a powerful antileukemic effect. But even in apparently perfectly matched donor-recipient pairs, some immune cells attack the patient’s normal tissues, causing potentially fatal GVH disease.

Because older leukemia patients are especially vulnerable–about half may die of GVH disease—the team targets this older cohort (whose average age is 64) for clinical trials of measures aimed at minimizing GVH reactions, while maximizing the allogeneic attack on leukemia cells that may have withstood chemotherapy.

In 14 patients thus far, the investigators have seen 100 percent transplant engraftment, with significant reduction in GVH disease and no deaths to date attributable to that cause.

The key to this GVH reduction in Barrett’s work, as well as in Bishop’s, has been to temper immune reactions by selectively eliminating the cells responsible for them—GVH-specific T cells. Studies with mice showed that low doses of fludarabine and cyclophosphamide effect a slow depletion of T cells in a transplant recipient, permitting engraftment of even HLA-mismatched cells without having to completely destroy the recipient’s immune system.

Working with patients with metastatic breast cancer, Bishop is employing chemotherapy regimens that gently, slowly deplete T cells as they kill breast cancer cells. Patients are left with disease-attacking natural killer (NK) and other immune cells, but free of the T cells’ destructive reactions.

Bishop says a key may be promoting Type 2 cytokine interactions that suppress GVH reactions, while squelching Type 1 reactions that promote them.

He emphasizes that hematopoietic stem cell therapy (HSCT) goals differ among patients. In some patients, the target of therapy may not be cancer but, rather, anemia and other nonmalignant blood diseases in which there is no benefit in retaining potential for graft-vs-tumor reactions.

Among cancer patients, the blood cells themselves may be cancerous or blood may harbor cancer cells. Similarly, T-cell numbers and reactivity may be naturally high or low.

"We don’t believe one regimen fits all," Bishop said He envisions blood stem-cell therapy will be pretty much "designer therapy."

Wrap-Up: (left to right) Cynthia Dunbar, senior clinical investigator, NHLBI; E. J. Read, chief of the Cell Processing Section, Department of Transfusion Medicine, CC; Betsy Nabel, director of clinical research programs, NHLBI; and Ron McKay, senior investigator, Laboratory of Molecular Biology, NINDS, lead a general discussion at the end of the daylong workshop on "Clinical Applications of Stem Cells at the NIH"

Array of Applications

Roland Martin of NINDS is examining the use of HSCT for a completely different purpose—stopping the decline of patients with multiple sclerosis (MS). Martin’s group shows that HSCT halts the progress of the autoimmune disease by eliminating MS patients’ immune cells that are programmed to attack their own myelin, causing inflammation and subsequent brain-cell loss. The patients’ immune systems are rescued with naive cells from transplanted hematopoietic stem cells.

In the seven patients treated thus far, this approach has reset the immunological clock, leaving patients free of the inflammatory component of MS.

Stopping the clock, however, doesn’t reverse neurodegeneration that has already occurred. For this, Martin says, one has to look at a second transplant of some other stem cells—oligodendrocyte precursor cells alone or with local delivery of growth factors. Transplantation of these precursors in mouse models of MS has restored myelinated cells and appropriate brain architecture, left no scarring, and, as a result, improved motor function.

Other research presentations in the center ring of the workshop featured a diverse array of results, ranging from NIDCR’s Pam Robey’s elaboration of stem cells in the dental pulp of children’s baby teeth to NIAID’s Harry Malech’s description of progress toward effective gene therapy for chronic granulomatous disease.

For example, Rocky Tuan of NIAMS described continuing refinements in a bag of innovative tricks designed to charm mesenchymal stem cells into making replacements for tissues damaged by arthritis and other ravages of age and degenerative joint disease.

Tuan has been inching ever closer to producing lab-grown tissues that closely mimic human bone and cartilage in texture, strength, ability to support appropriate growth and differentiation of cells, and even transmission of critical signals for cell-cell interactions.

Tuan noted that understanding these signals—and learning how to control them—may someday obviate the need for transplants of stem cells or lab-grown tissues.

The ideal course would be to reprogram cells or call in residual mesenchymal stem cells already within the body to repair damaged joints. Look at the salamander’s ability to regrow complete limbs, Tuan urges. "It does all these things without any government intervention or support!"

Do Reparative Cells Come from Near or Far?

One key question for scientists working with some adult stem cells is where the cells actually come from. Do repairing cells already reside in tissues and multiply when needed to repair adjacent damage? Or are the cells summoned from a stem-cell source elsewhere in the body?

NIDDK’S David Harlan and Nadya Lumelsky say that their studies aimed at diabetes suggest that insulin-producing b-cells may not arise, at least not to date with clinically relevant efficiency, from circulating stem cells or other pancreatic cells. Harlan presented work that did not support the existence of bone-marrow resident stem cells capable of differentiating into insulin-producing cells.

In other work with human pancreatic cells, Lumelsky had variable success in cultivating new insulin-producing cells in comparatively short-term cultures. The success of the culture varied greatly depending on the specific human islet isolation.

That said, her laboratory takes cellular clusters from the disaggregated human pancreas and grows cells from the mixture on fibronectin in defined culture medium (with a cocktail of growth factors), promoting cellular re-aggregation.

With this system, Lumelsky finds that a minority of such preparations make more C-peptide (a marker for insulin production) than what she started with, while most make less.

Evidence of remote sources of stem cells came from NEI’s Karl Csaky, who presented data on bone marrow as one key source of cells involved in neovascularization in the eye.

Csaky’s mouse studies on ocular overexpression of VEG-F or laser-induced ocular damage showed that 70 percent of macrophages, 58 percent of endothelial cells, and 60 percent of smooth muscle cells involved in neovascularization and repair came from the bone marrow.

In contrast, none of the retinal cells in the repaired tissue came from the bone marrow. Csaky is interested in identifying factors responsible for summoning and triggering appropriate differentiation of stem cells at sites of damage in the eye.

The Heart of the Matter

Focusing on heart repair, Richard Cannon of NHLBI recalled studies from 1997 that detected endothelial progenitor cells in peripheral blood and evidence from animal models of hind-limb ischemia that injection of these cells can stimulate growth of new blood vessels and improve blood flow to the affected extremity.

Attempts to use such cells to revascularize human hearts suggest benefit to myocardial blood flow and function, but the nonrandomized clinical trials reported to date have involved small numbers of patients who often underwent coronary revascularization at the time of treatment.

Studies performed in the NHLBI Cardiovascular Branch suggest that there may be variation in the ability of people’s stem cells to accomplish the feat of repairing heart damage.

The researchers isolated mononuclear cells from blood samples of middle-aged men with varying levels of risk for heart disease and examined the ability of progenitor cells to produce endothelial-like cells needed to form new vessels in damaged hearts.

Cells from subjects with the fewest risk factors produced the most colonies of reparative cells, and those with the greatest number of risk factors had the lowest numbers of functional colonies.

Further, endothelial function, as tested by nitric oxide–mediated vasodilation, correlated strongly with subjects’ endothelial colony-forming capacity. The researchers speculate that loss of the cells’ ability to repair endothelial damage in the heart may be yet another independent contributing factor in cardiovascular disease.

Cannon and his colleagues studied whether just mobilizing stem cells from bone marrow with the cytokine granulocyte–colony-stimulating factor (G-CSF) might be sufficient to help repair hearts damaged by severe cardiovascular disease. Although they were successful in augmenting endothelial progenitor cells in the circulation after G-CSF administration, the numbers of cells were small, the duration of mobilization short, and the benefits to patient health undetectable on cardiac MRI and treadmill stress tests.

Cannon will soon be trying direct myocardial administration of stem cells to patients’ damaged hearts.

In a collaborative study with Suburban Hospital’s (Bethesda, Md.) Cardiopulmonary Rehabilitation Unit, Cannon is exploring interrelationships of exercise, endothelial function, endothelial progenitor cell mobilization, and nitric oxide bioactivity in hopes of giving heart patients a vascular repair profile more like that of healthy individuals.

More Tools for the Trade

Still other NIH investigators are developing tools and facilities to aid stem-cell research. New tools include in vivo imaging and clinical-grade reagents that permit investigators to "see" stem cells at work inside the human body.

Robert Lederman of NHLBI described sophisticated MRI techniques that could be used to watch precise delivery of bone marrow stromal cells to the margins of an infarct, for example, or to calculate peripheral vascular function by measuring reperfusion of blood into tissue when a restraint on circulation is abruptly removed.

"These images are incredibly sensitive tools," Lederman finds. But frustrations in working with industry partners to develop the techniques have led Lederman to agree with Barrett that NIH needs a "transitional" core. If there were one, "I’d be discussing results rather than experimental design," Lederman said.

Lederman says one key to his work is a technique for magnetically labeling cells developed by the CC’s Laboratory of Diagnostic Radiology Research. Joe Frank, who leads LDRR, described ongoing development of the superparamagnetic ironoxides that are taken up by cultured stem cells, for example.

As few as 50,000 labeled cells can be injected into a living animal and then observed via 1.5 Tesla MRI—a field strength that could also be used in human studies.

Frank says nondividing cells, such as T cells, retain label for 40 to 60 days, and there is no change in apoptosis or reactive oxygen species in labeled cells. As paperwork wends its way through the FDA to get the magnetic label approved for clinical use, Frank has been collaborating on studies that put the vital stain to work to study angiogenesis, stroke, MS, and cardiovascular disease.

The Challenges of Product Development

If NIH wanted to take up Barrett’s call for a transitional research core, perhaps the best advice on such a facility would come from E. J. Read, chief of the Cell Processing Section of the CC Department of Transfusion Medicine.

"Product development," Read says categorically, "is different from basic science." Product development studies aim to design cellular products that meet the needs of the clinical trial as well as FDA’s regulatory requirements. The new products must ultimately be prepared in a GMP environment to ensure that they are safe and effective for patients who receive them.

Translation of cell therapies requires working collaboratively with investigators, starting in the preclinical phase, to define and characterize specific details of each product.

Challenges abound. The most striking, Read says, is the variability of cells–starting material from different patients. This variability means contingency planning is critical because things will go wrong. Scale-up and movement from lab to clinic may not be straightforward. Closed, automated systems are more desirable, she said, because they reduce human error and the risk of contamination from microbes in the environment when cells are processed.

Collaboration with industry brings in lots of additional challenges. Read says her best allies in coping with FDA oversight have been the facility’s masterfile of operational and quality procedures, written in responses to FDA’s formal queries and published guidelines, and copious documentation during the preparation of each clinical product. "This has really helped a lot," Read says.

NINDS’ Ron McKay gave the workshop a view on another core facility with an update on the NIH Human Embryonic Stem Cell Unit (see The NIH Catalyst, March-April 2004).

That facility is growing and characterizing human embryo stem cells, "currently the most useful" stem cell of all, by virtue of its capacity "for infinite expansion to lots of cell types," McKay said. "Somatic stem cells are not designed for infinite expansion."

McKay says studies of embryo stem cells in rats show that they can be differentiated into dopamine-secreting neural precursor cells and implanted into the ventral midbrain, where they engraft beautifully and are electrophysiologically and behaviorally functional.

"In five years," McKay predicted, "human embryo stem cells will routinely be put to many clinically relevant uses."

Regulatory Hurdles

Whether the prediction is borne out depends in part on reasonably swift approval of stem-cell procedures by the FDA. FDA’s Steve Bauer acknowledged that stem-cell–based therapy is a relatively new regulatory area for the agency, but he noted that development of specifications for stem-cell–based products is evolving rapidly as scientific information increases.

The agency will be looking not only at the end products but also at initial starting materials, culture, and other processing of cells. Bauer said key issues might include:

Potency of cells

Freedom of starting materials from infectious disease

Derivation of the cells

Stability of the cells

Propagation conditions

Characterization of the cells

Where implanted cells go and how long they live

Tumorigenicity of implanted cells

Ectopic development of tissue from implanted cells

In a panel discussion concluding the workshop, Barrett said the NIH intramural program has a unique opportunity in stem-cell research. "We have all the pieces in one place and lots of expertise to see the research through to clinical trials." NHLBI Clinical Director Betsy Nabel concurred, citing the workshop’s display of "incredible breadth and depth, from basic to applied research."

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