By David N. Leff

Blood is thicker than water - not just proverbially but physically. Try this simple experiment: Place a drop of blood and a drop of H₂O side by side on a dish. The water evaporates completely; the blood leaves a stain behind.

In actuality, the water's specific gravity (by definition) is 1.00; that of blood, 1.06. What accounts for the thickness is a series of life-preserving proteins. Red blood cells nourish the body with oxygen and nutrients; white blood cells defend it with an immune arsenal of antibody-generating B cells, protective T lymphocytes, natural killer cells, as well as wound-sealing platelets. Not to mention monocytes, and granulocytes.

Where do all these vital blood products come from? To begin with, hematopoietic stem cells in the body's bone marrow beget progenitor cells. These in turn beget the panoply of terminal blood cells.

Listen to hematologist and biochemist Cesare Peschle of Thomas Jefferson University in Philadelphia: "The pluripotent stem cell is the lifetime source of all blood cells, because it has a very extensive self-replicating capacity. On the one hand it maintains itself; on the other hand, it differentiates to progenitor cells and then up to terminal cells. Progenitors," he went on, "are immature cells, with little or no self-renewal capacity. After they differentiate, they just extinguish themselves."

Peschle recalled, "In the journal Science 10 years ago we pioneered purification of progenitors. To do so we utilized the CD34 marker, which is expressed on the outer membrane of all progenitor cells. Then we went into trying to refine stem cells, which are very rare. There are just a very few of them within the progenitors. It has been calculated," he pointed out, "that there are approximately one in a 100,000 stem cells in bone marrow; one per 1,000 progenitors.

"So when we found it difficult to isolate them from the purified progenitors," Peschle recounted, "we soon understood that it would be almost impossible to do it unless we had a marker, more or less specific for the stem cell, comparable to the CD34 markers for progenitors."

In 1994, Peschle and his co-workers found their purified progenitors expressed a very low level of vascular endothelial growth factor receptor (VEGF-R). This molecule also goes by an alias named KDR - an acronym of uncertain origin.

"We were surprised," Peschle said, "because KDR - the synonym for VEGF-R - was linked only to endothelial vascular cells, as the name implies. So we said: 'What the hell does KDR have to do with blood progenitors?' Then we realized that very early in embryonic life, primitive endothelial cells are produced in very close - and possibly functional - contact with primitive hematopoietic cells. And we reasoned that since embryonic endothelial cells are KDR-plus, possibly primitive stem cells could be KDR-plus, too."

Job One: Find KDR-Parsing Antibody

To pursue this line of reasoning, he and his group had first of all to find an antibody that would purify KDR-plus from KDR-minus cells, within the population of the CD34-positive progenitors. They located a high-affinity antibody against KDR's extracellular domain, and used it to characterize the CD34-positive progenitors. They found that less than 0.5 percent of them carried KDR on their surface. All the rest were KDR-minus.

Peschle wears two hats. He heads the laboratory of microbiology and immunology at Jefferson, and chairs the department of hematology-oncology at the Higher Institute of Health in Rome. He is senior author of a paper in the current Science, dated Sept. 3, 1999, titled: "KDR Receptor: A key marker defining hematopoietic stem cells."

"The hematopoietic stem cell," Peschle told BioWorld Today, "has been considered the elusive Holy Grail of hematology and immunology. Now it has been found and captured by identifying the first specific and functional stem cell marker."

After preliminary in vitro assays, he and his co-authors nailed down the evasive stem cell in vivo - first in mice, then in fetal sheep in utero.

"We sub-lethally irradiated immunodeficient NOD-SCID mice," Peschle recounted, "to wipe out their entire blood-forming system, and then injected the human bone-marrow KDR cells to be tested for stem cell activity. If they were present," he explained, "they were going to repopulate all of the mouse's hematopoietic site. The animals were killed after three months, and checked for human cells of various erythroid, lymphoid and myeloid blood-forming types

"The other in vivo test we utilized," Peschle continued, "was uniquely performed by E.D. Zanjani, a co-author. He did the pre-immune fetal-sheep assay, by injecting the cells into the fetuses of eight pregnant ewes, still in the uterus. Then he checked, after two months, for human cells repopulating the fetal sheep's hematopoietic tissues.

"In both cases we found - and we were very astonished - that all repopulating stem cells were on the KDR-plus fraction, and none was present in the KDR-minus one. Progenitors were present in the KDR-minus fraction; stem cells were all in the KDR-plus fraction - which is less than 0.5 percent."

Blood Transfusion By Stem Cell Donor

Peschle's Science paper concludes: "These issues are of pivotal importance for a large array of biotechnological and clinical challenges, such as hematopoietic stem cell transplantation, in vivo blood cell generation for transfusion and hematopoietic gene therapy."

He observed, "I think these three biotechnology and clinically significant goals are not remote. We still need time of course to develop those things, but in my view they are neither far-fetched nor in the very long time frame. All are in the next decade, perhaps even the next five years."

He sees blood transfusion as the most imminent application. "Stem cells produce an enormous amount of blood cells during our lifetime," Peschle said. "What we need to do is make them replicate in the laboratory their function in the body, then differentiate very extensively down to the terminal blood cells, which would be utilized for clinical transfusions.

"Biotech people may be interested to know," Peschle pointed out, "that all the KDR isolation procedure has been patented, so it's open potentially to biotech applications. And of course Thomas Jefferson University, and also the Italian institution, which is in part involved, would certainly consider contacts from biotech companies," he concluded, "to help solve these problems that we still encounter is applying those isolated stem cells to the three goals we mentioned."