College of American Pathologists
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Beyond blood cells: stem cells’ startling potential

July 2002
Vida Foubister

A recent stem cell study, considered revolutionary by those in the field, found that stem cells circulating in human peripheral blood can make cells of the liver, gastrointestinal tract, and skin.

These novel findings, which challenge traditional thinking about tissue repair, are based on data derived from a sophisticated double-staining technique that combines fluorescence in situ hybridization with immunofluorescent microscopy.

"Up until recently, we thought that hematopoietic stem cells in peripheral blood make blood cells and that’s it," says Martin Körbling, MD, professor of medicine and clinical director of apheresis in the Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Houston, which conducted the study. "Now the thinking is much more dynamic. Adult stem cells can not only make blood cells, they can make liver cells, gut cells, and skin cells."

Unlike in embryos, adult stem cells have long been thought to be programmed to make just one type of cell based on where they are located. The work of Dr. Körbling and his colleagues, published in the March 7 issue of the New England Journal of Medicine, is the latest to suggest that adult stem cells have tremendous versatility. Previous studies on stem cells found in bone marrow, for example, have shown that they not only make new blood cells but can make hepatic oval cells, hepatocytes, cholangiocytes, skeletal-muscle cells, astrocytes, and neurons.

"There is plasticity in the system," says Zeev Estrov, MD, professor of medicine and director of hematopoiesis research in the Department of Bioimmunotherapy at the M.D. Anderson Cancer Center. "One type of stem cell can become different tissues. These are new and exciting concepts."

This work has led to the hypothesis that adult stem cells in the peripheral blood, in addition to cells at the site of tissue injury, are involved in repairing tissue damage.

These findings offer hope that stem cells circulating in the bloodstream could be used "in the future, perhaps in the near future" to repair tissue damage in organs such as the heart or central nervous system or even to replace diseased tissue, says Dr. Estrov.

Study details

Given that bone marrow stem cells may become mature cells of various organs and that such cells transit the peripheral blood, the investigators set out to determine whether circulating stem cells have a similar potential. (Unlike bone marrow stem cells, circulating stem cells easily can be collected from the blood using apheresis.)

To test their hypothesis, they used the Y chromosome as a marker to trace the pathway of peripheral blood stem cells that were transplanted in six female patients from male siblings. Of the six control patients, five received sex-matched stem cell transplants and one received an autologous transplant.

Ruth L. Katz, MD, professor of pathology at the M.D. Anderson Cancer Center and a principal investigator in the study, analyzed biopsy specimens of the skin, liver, and gut for chimeric cells. This required her to demonstrate the presence of male donor cells in female organ tissues and to show that these cells were epithelial in nature and not hematopoietic.

The tool Dr. Katz used, called FICTION, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms, combines two common laboratory techniques: immunofluorescence for detecting antigens in tissues and interphase FISH for detecting X and Y chromosomes. Using this technique, hundreds of cells can be rapidly screened for rare events or cell-to-cell heterogeneity.

Initial attempts to analyze autopsy tissue failed. "These cases were very old," says Dr. Katz, who is also chief of research cytopathology and director of image analysis at the M.D. Anderson Cancer Center. "They were fixed in formalin, and there probably was a lot of cross-linking of proteins. With FISH, you have to digest tissues to get rid of exogenous proteins and RNA, and you want to make sure to get a good signal-to-probe ratio."

With biopsy tissue, however, "we got it to work beautifully," she says. Cells were identified as epithelial in nature through staining with fluorescent cytokeratin antibodies, and their location on an automated motorized microscope stage was recorded. The slides were then stained with centromeric probes for X and Y chromosomes, and the same cells that expressed cytokeratin were screened for the expression of X and Y chromosomes.

The investigators found that up to seven percent of the cells in the female patients’ liver, skin, and gut tissue had become chimeric. XY- and cytokeratin-positive cells were detected in liver tissue as early as day 13 and in skin tissue as late as day 354 after transplantation. "From a pathologist point-of-view, the pictures are incredible," Dr. Katz says. "There are cells with XX chromosomes and then you see a cell with XY chromosomes. It’s very dramatic".

As expected, no XY chromosome-positive cells were detected in the three female patients who received a stem cell transplant from female donors. Male patients transplanted with cells from a male donor had XY-positive cells in up to 75 percent of their biopsy cell specimens. Consistent with the literature, XY-positive cells were detected in less than 100 percent of these cell specimens for technical reasons, including incomplete sections of nuclei and stringent counting criteria.

Immunohistochemistry was performed on consecutive sections of tissue to demonstrate that the XY-positive, donor-derived cells were tissue-specific cells. Slides with matching microscopic fields were stained with hematoxylin and eosin and a CD45 antibody to exclude the presence of lymphocytes, monocytes, and granulocytes.

The organ specificity of the cells was indicated by their location, staining for cytokeratins in skin and gut biopsies and hepatocytes in the liver, and the absence of CD45. XY-positive hepatocytes in the liver were distinguished by large, round nuclei and abundant granular cytoplasm that was also positive by Texas Red-tagged cytokeratin. The donor-derived cells in the epidermal tissue of the skin were located in the deep layer of Malpighi, close to the dermal-epidermal junction and the stratum granulosum. In the glandular epithelium of the gastric cardia, cells containing the Y chromosome were found in the foveoleae or tubular pits of the superficial glandular layer.

Tissue repair

Because the double-staining FICTION procedure can negatively affect the integrity of tissues, it might have led the investigators to underestimate the number of donor-derived, XY-positive cells in female recipients. Recent studies have found that under certain conditions, the number of donor-derived cells can be as high as 40 to 50 percent, compared to zero to seven percent in this report.

"If you induce chemotherapy-related tissue damage or there is liver cirrhosis or liver failure conditions, then you would probably need more cells to repair and recover that tissue," Dr. Körbling says.

It is likely that two factors are necessary for tissue repair: stem cells at the site of injury and signals triggered by tissue damage from high-dose radiotherapy or chemotherapy and graft-versus-host disease following stem cell transplantation that recruit peripheral blood cells to regenerate the tissue. "The more circulating stem cells you have available, the better the tissue repair," Dr. Körbling says.

Engraftment of donor-derived hepatocytes and epithelial cells in the current study was uniform, however, irrespective of the presence or absence of tissue damage caused by graft-versus-host disease.

It is not known where these circulating stem cells originate or how they generate hepatocytes and epithelial cells. One explanation is that each tissue has its own circulating stem cells. Another is that primitive adult multipotent stem cells may give rise to differentiated, lineage-restricted stem cells that can generate mature cells. Stem cells committed to a differentiation pathway might also be able to switch lineages under specific conditions. It’s also possible that differentiated cells can dedifferentiate into cells with multiple capabilities.

Though it’s still "space agey and hypothetical," Dr. Katz says, pathologists might play a role in analyzing the regenerative capacity of stem cells. "In the future, stem cells might have the potential to go in and repair organs. For instance, they’ve shown in patients who have myocardial infarctions that stem cells can go in and turn into cardiomyocytes and help repair the heart," she says.

"If we start using stem cells therapeutically," Dr. Katz adds, "it’s possible that we’d be able to use FISH to work out exactly how complete the repair had been."

Several research groups in the United States and Western Europe are testing the potential of peripheral blood and bone marrow-derived stem cells to repair damage in other organs, says Dr. Estrov. Because these stem cells come from adult donors, this work sidesteps the political debate over the use of embryonic stem cells.

Human blastocysts, which must be destroyed to produce embryonic stem cell lines, are considered by some to be embryos that should be accorded full human rights. Those who take this view oppose the destruction or biopsy of blastocysts, which they believe constitutes the taking of lives, to create embryonic stem cell lines. Others, however, including many patient and research groups, support this research because they believe it may lead to treatments or cures for many diseases, including Parkinson’s disease, diabetes, heart disease, multiple sclerosis, burns, and spinal cord injuries.

While the current study focused entirely on adult stem cells, the investigators believe both embryonic and adult stem cell research should be pursued. "The question that is often asked is, ’What is the better strategy?’" says Dr. Körbling. "The short answer is there is no comparative data available. There is no way, right now, to compare the potential of embryonic stem cells versus adult stem cells."

Vida Foubister is a writer in Mamaroneck, NY.