Adult human stem cells may offer the opportunity to use one of biomedical science's most promising technologies without the ethical dilemmas of embryonic cells. But whether the cells' plasticity-or ability to ignore germ-line heritage and differentiate into therapeutically useful tissues-warrants clinical application at this stage remains controversial.
"We're still debating it," says Amy Wagers, Harvard Medical School investigator and plasticity critic. "It's too early to tell which way things will fall." Biologists generally agree that even the most potent adult stem cells can't approach the therapeutic power of embryonic stem cells. Nevertheless, at least a dozen clinical trials based on adult-cell plasticity have commenced in patients with serious heart disease – prematurely, some contend.
In embryonic development, cells form three germ layers: ectoderm, mesoderm, or endoderm. Generally, biologists considered cell differentiation overwhelmingly unidirectional and progressively restrictive. A cell fated to make neurons could not make blood cells; a stem cell fated to make white and red blood cells could not make a heart or a liver. Somatic cells could not transdifferentiate, switching from one lineage to the next; nor could they dedifferentiate, reverting to less specialized versions.
Research since the late 1990s argued that some stem cells are significantly more flexible than originally thought. A flurry of animal research hinted that hematopoietic stem cells (HSCs) could become neural, muscular, skeletal, liver, kidney, lung, and skin cells. HSCs went to the site of the injury and apparently changed into a mélange of tissues. In one influential paper, Donald Orlic at the US National Institutes of Health reported that massive numbers of HSCs had changed into cardiomyocytes in heart-injured mice.1
Changing Cells or Changing Arguments?
Basic scientists continue to dispute whether truly plastic cells can be found in adult cell populations. Yale University professor Diane Krause says she believes there is a potent stem cell in the bone marrow that can change into lung, liver, and skin.1 She rejects that hematopoietic stem cells (HSCs) transdifferentiate, however, and instead says she has found a yet-uncharacterized "marrow-derived cell." According to Krause, "There is no question that a marrow-derived cell can make epithelial cells. The question is how: fusion or differentiation. I feel it could be both."
Johns Hopkins University oncologist Saul Sharkis, who collaborated with Krause in 2001, rules out fusion. "The most important thing is to know what kind of cell you transplant. A primitive cell has more potential."
In 2002, the University of Minnesota's Catherine Verfaillie provided evidence for multipotent adult progenitor cells (MAPCs).2 MAPCs are extremely rare – numbering less than 2,000 in a single mouse. They live indefinitely in culture and can be coaxed to generate different germ layers and liver, neural, and endothelial cells. The difficulty with MAPCs is that they haven't been identified in vivo and have a finicky nature, making Verfaillie's experiments hard to repeat.
Scott Dylla, a scientist with OncoMed Pharmaceuticals, spent five years working with Verfaillie. Dylla says though MAPCs were thought to be HSC "changelings" early on, Verfaillie was more careful than most about genetically marking clonal populations before observing their differentiation pathways.
Better characterization of the starting material could help. Before transplanting stem cells, Harvard Medical School's Amy Wagers uses fluorescence-activated cell sorting to pick out pure HSCs on the basis of 12 surface antigens, including the important CD45 markers. For her part, Krause purifies cells taken from bone marrow, though she admits some heterogeneity. Sharkis first elutes cells based on size and density, and then removes nonstem cells with an antibody (a step called lineage-depletion). He labels the remaining cells with a dye and puts them back into an irradiated mouse. The damaged marrow swings into a repair mode, enriching the cells 1000-fold. The marrow is harvested and transplanted, repairing damaged organs.
Sharkis and Krause may indeed have their hands on a rare MAPC-like cell. But Wagers sees no differentiation of her cells into heart or lung. Krause says Wagers' purification scheme could leave behind a new "marrow-derived" cell. And Dylla posits that the claims of new potent adult stem cells found in blood could be "rediscovered" MAPCs. The round robin of claims and counter claims are evidence of a subtle – but important – shift in the debate about stem cell plasticity. At least among this group of hematopoietic stem cell experts, the discussion has turned to whether novel varieties of multipotent stem cells exist in the bone marrow.
The relative safety of injecting heart patients with their own mobilized blood prompted a clinical trial in Brazil, and by 2003, ten international trials had enrolled human subjects with end-stage heart disease. Clinicians reported anecdotal cases of formerly bedridden patients jogging after the procedure.
In early 2004, the US Food and Drug Administration reviewed the European data and approved similar trials in Boston, Texas, and most recently, at Johns Hopkins University in Baltimore. The Baltimore studies use powerful mesenchymal cells, a multipotent stem cell found in bone marrow. Nevertheless, the European trials showed modest improvement in heart function after myocardial infarction, along with some evidence of angiogenesis. Left ventricular ejection fraction, the percentage of blood pumped into the aorta, improved as much as 6% more for treated patients than for controls. Others showed no significant differences in improvement.2
Douglas Losordo, a Tufts University clinician who has treated his patients with HSCs, says the therapy isn't designed as a cure but is akin to "giving a booster dose of the natural mechanism for tissue repair." Robert Lanza, vice president of medical and scientific development at Advanced Cell Technology in Worcester, Mass., says he's not surprised by the early clinical data. He reports that in his mouse research, blood progenitor cells replaced nearly 40% of damaged heart tissue. Lanza maintains, "As long as the HSC-heart trials are done safely, the results give us good information." He adds, "If I had a heart infarct, I'd be happy with a 10% improvement."
Critics point out that the initial trials were conducted with few patients and note potential flaws in the conclusion that hematopoietic cells changed into heart muscle cells. Stanford Nobel laureate and biochemist Paul Berg says, "The mouse studies used unpurified populations of stem cells. So did the heart trials. It could be some growth factor carried along with the supernatant or a resident heart stem cell that's responsible for the effect, not plasticity."
By early 2004, many of the original claims of stem cell switching had been refuted. Early results couldn't be repeated and experimental design was called into question. Several papers indicated that adult stem cells had not changed into, but rather fused with organ cells. Two labs attempted to repeat experiments showing blood-heart plasticity in mice and primates, but failed.34
In a follow-up study, Wagers, along with Stanford's Leora Balsam, Robert Robbins, and HSC pioneer Irving Weissman, injected highly purified populations of genetically tagged HSCs into the heart muscle of 23 mice. The transplanted cells did not increase the survival rate in mice, did not typically persist in the heart muscle more than 30 days, and did not produce the signature proteins of heart cells.5 They did notice slightly improved pumping efficiency, however.
It's important to note that the bone marrow-heart trials have been conducted safely, and the clinical effect, while small, is significant. Despite this, many basic scientists say the clinicians moved too swiftly. Transplanting mixed populations of cells, they say, leaves the mechanisms of the therapy locked in a black box. "It is important to do as much basic research as we can before going to clinical trials," says Johns Hopkins University professor Saul Sharkis. "We still don't completely understand the mechanisms of stem cell repair."
Wagers worries about declaring victory too early. "If we consider a 6% improvement in cardiac function a success, then we've left behind an opportunity to understand why this is happening and aim for a 60% improvement." Clinicians bristle when told that they should slow down. Joshua Hare, principal investigator for the Johns Hopkins trial says, "It's unethical to wait. We won't fully understand the mechanism until we do the clinical research. That's what evidence-based medicine is all about."