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Argonne technologies win R&D 100 Awards
Serendipity in lab turns blood into stem cells Cutting blood supply to tumors could stop them Argonne Update
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Serendipity in lab turns
blood into stem cells
Continued ...
Making stem cells from blood
Using blood from LifeSource Blood Services blood bank, which was taken from a variety of healthy donors, the researchers systematically inoculated the cells into culture plates.
Usually, 20 million white blood cells were inoculated into a growth medium that contained vitamins, amino acids, serum, antibiotics, salts and a bicarbonate buffering system. Dishes were incubated for eight to 12 hours at body temperature, 98.6 degrees Fahrenheit.
 'PLURIPOTENT' CELLS — This micrograph shows a colony of undifferentiated human embyonic stem cells. These "pluripotent" stem cells are able to morph into a variety of cell types. Argonne researchers have developed an easy-to-replicate process to create these cells from human blood. Image courtesy of University of Wisconsin-Madison. |
After rinsing the dishes with fresh growth medium, the adherent cells were detached using forceful pippetting — a process that uses medium to remove cells from dishes. The detached cells were identified by immunostaining to verify that they were monocytes. Immunostaining identifies cell types using antibodies to detect and label antigens — proteins capable of stimulating an immune response. The antibodies are labeled with fluorescent markers so they can be seen under a special microscope.
Through immunostaining, the biologists determined that the cells they had separated were monocytes, the type of cells needed for their experiments. They incubated the monocytes with macrophage-colony-stimulating factor and found that after five days the cultures morphed into two types of macrophages.
While most were the usual, round-shaped macrophages they expected to find, others were elongated and resembled fibroblasts — cells that make up connective tissue. While these cells displayed the typical macrophage markers, they also exhibited elevated levels of a blood cell-forming marker, indicating they were blood stem cells.
Huberman, Zhao and Glesne concluded that the fibroblast macrophages were the cells they accidentally discovered in their initial experiment and these would behave as stem cells. But were they pluripotent? Could these cells be made to form a variety of other cell types?
Proving cells pluripotent
The researchers showed that fibroblast macrophages originating from monocytes could grow into blood cells other than macrophages. Adding Interleukin-2, also known as T-cell growth factor, to their fibroblast macrophages, the biologists found the resulting cells expressed characteristics of T-lymphocytes - blood cells that participate in the immune response.
The biologists now knew that cultured fibroblast macrophages could form other blood cell types. They delved further into experiments to see what other cell types fibroblast macrophages could grow into.
They found they could induce the fibroblast macrophages to differentiate into cells with markers — indicators — for skin, liver, blood vessel and nerve cells.
Applying epithelial growth factor resulted in cells that displayed properties of epithelial cells — the cells that make up the skin and inner linings of the lungs and digestive tract. Similarly, endothelial growth factor caused the fibroblast macrophages to form cells with markers for blood vessel cells.
Further tests showed that using nerve growth factor caused the fibroblast macrophages to differentiate into cells that, among other properties, had neurite and axon-like processes, protrusions indicating that they would behave as nerve cells.
Mature tissue issue
 SERENDIPITOUS SCIENCE — Biologists Eliezer Huberman (right) and Yong Zhao investigated what was left in the culture plates Zhao left unattended during an illness. What they found led them to discover the human blood stream is a source for stem cells. |
Last year, a study conducted at the University of Minnesota Medical School showed that adult stem cells extracted from bone marrow had pluripotent characteristics. When injected into mice such cells developed into blood, liver, lung and intestinal cells. Some scientists said, however, that adult stem cells differentiate into other cell types as a result of fusion with preexisting mature tissue cells.
Argonne's biologists proved that fibroblast macrophages could differentiate into cells belonging to a variety of cell lineages without such a fusion.
To prove this point, they inoculated a small number of monocytes into tissue culture plates containing separate wells and inspected these under a light microscope to be sure that only wells containing single cells were included in the experiments. The progeny of these single cells, which did not contain preexisting mature tissue cells, also morphed into T-lymphocytes, epithelial, blood vessel, liver and nerve cells. Based on these experiments, the biologists concluded that mature tissue did not need to be present for fibroblast macrophages to differentiate into other types of cells.
Putting stem cells to work
Pluripotent stem cells may be used in transplants in the same patient, called autologous transplantation therapy. Doctors would collect a patient's own blood cells and induce them to grow into the tissue desired for treatment. For example, if the patient had a damaged liver, the doctors would apply hepatocyte growth factor to the patient's blood cells. These healthy cells would be transplanted into the liver to generate healthy tissue and repair the damage.
But a good storage method is needed to keep a plentiful supply of these potential healers. The researchers tested cells stored in liquid nitrogen to determine if storage would have any effect on differentiation. They found the liquid nitrogen had no effect on differentiation later, demonstrating it to be a viable storage method. Doctors could keep stem cells on hand for later treatments.
From stems to studies
From the monocyte-generated stem cells, Huberman says that he hopes to extend his research to induce cells to differentiate into insulin-producing cells, which could be used to treat diabetes.
Huberman would also like to focus on developing treatments for neurological disorders. He plans to inject differentiated cells formed from monocytes into mice to determine this treatment's effectiveness.
"I'm very excited about this research," says Huberman. "Our procedure is simple so that many labs will be able to repeat and extend the experiments, and as such I hope it will mean progress in the use of our type of stem cells to study and treat human diseases."
For more information, please contact Catherine Foster (630/252-5580 or cfoster@anl.gov) at Argonne.
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