by Catherine Shaffer
A major hurdle in stem cell research has been identifying and characterizing stem cells in cultures and tissues. Because these cells are largely indistinguishable from their progeny, and stem cell separation methods typically include progenitor cells as well as stem cells, researchers must use indirect methods to identify and isolate them.
Stem cells in blood and bone marrow
Hematopoietic stem cells are some of the most extensively studied stem cells, and many tools and techniques have been developed for working with these cells. Often, methods first developed for blood cells have been adapted to other cell types. Although immunological methods promise to simplify identification and isolation of hematopoietic stem cells, the transplantation assay continues to be the standard by which all others are measured. The assay can be qualitative, simply giving information on the identity of the cells in question; or it can be quantitative, comparing transplanted mice with different numbers of cells. For example, one mouse might receive ten cells, another one hundred thousand. Several months later, the number of mature blood cells relates to the original graft, creating a dose-response curve. The transplantation assay can also be used to compare unfractionated bone marrow to enriched bone marrow, or unmodified stem cells to cultured stem cells. Selection is typically carried out using mouse strains that express particular white blood cell antigens, such as CD45.1 and CD45.2. If a CD45.1 mouse is transplanted with stem cells from the CD45.2 mouse, then the new blood cells should have CD45.2 antigen. The assay is applicable to human cells by using immune-deficient mice.
Transplantation is time consuming and expensive, however, so newer technologies in stem cell identification focus on immunological methods for identifying stem cells quickly in culture.
Identification methods
Stem Cell Technologies, a leading vendor of tools for stem cell research, offers a large catalog of antibodies, reagents, and other products for stem cell identification and isolation. One of their newest offerings is an antibody for endothelial protein C receptor (EPCR) for the identification of mouse hematopoietic stem cells, based on work done by Richard Mulligan et al. at Harvard Medical School, showing that mouse bone marrow cells isolated on the basis of EPCR expression were highly enriched for hematopoietic stem cell activity. That makes EPCR the first explicit antigenic identifier of hematopoietic stem cells in mice—and a possible workaround for the time-consuming transplantation assay.
Another Stem Cell Technologies method for identifying stem cells is the Aldefluor assay, for human hematopoietic stem cells. It uses the aldehyde dehydrogenase substrate BAAA as a marker. In stem cells, high levels of aldehyde dehydrogenase activity convert BAAA to the fluorescent molecule BAA. The cells can then be separated using a flow cytometer.
A major thrust of product development at Stem Cell Technologies is to support research on human therapies based on hematopoietic stem cells, and address gaps in knowledge that hinder the progress of new therapies. Bert Wognum, PhD, a senior scientist at Stem Cell Technologies, says: “The number of stem cells you have in a cord blood sample is not enough to regenerate blood cells in an adult patient. Clinicians are interested in ways to expand stem cells in culture.”
R&D Systems offers a series of functional identification kits for mesenchymal and neural stem cells of mouse or human origin. The mesenchymal stem cell kit identifies mesenchymal stem cells from bone marrow by their ability to differentiate into mesenchymal lineages. The kit includes reagents, media, and antibodies to differentiate adipocytes, chondrocytes, and osteocytes. The neural stem cell identification kit is similar, except that its antibodies select neural precursors, astrocytes, neurons, and oligodendrocytes.
According to Electra Coucouvanis, manager of the stem cell and developmental biology group at R&D Systems, the company produces its antibodies using full-length recombinant proteins, not peptides, which maximize specificity and signal strength. Says Coucouvanis: “Many stem cell populations are incompletely characterized with respect to marker expression, and can only be definitively identified by their ability to differentiate into particular lineages. Our functional identification kits allow a functional analysis of cell populations of interest and facilitate identification of stem cells.”
Isolation methods
Isolating stem cells from cultures once they have been identified presents a new set of challenges. Typically stem cells occur very rarely, so sorting them directly on a flow cytometer is a very labor-intensive prospect. This is where the same immunological technique used to identify the cells can become part of the system for isolating the stem cells from the culture. Magnetic separation has become very popular for stem cell applications. BD Biosciences' Imag system uses a magnetic particle between 0.1 and 0.45 uM in size, coated with monoclonal antibodies. Antibodies available for the system include antibodies for T cells, lymphocytes, hematopoietic progenitor cells, and a large selection of surface antigens such as CD4. Samples that have been enriched using the Imag system can then be sorted further by flow cytometry. The small magnetic particles don't interfere with further growth of the cells, so they can actually be used in culture after sorting.
Stem Cell Technologies also offers an immunomagnetic cell separation system. EasySep is a column-free method for all cell types, and StemSep is a column-based method for human stem cells; these can be used for positive or negative selection of cells. For negative cell selection only, they offer RosetteSep, for human cells, and SpinSep, for human and mouse cells. RosetteSep takes advantage of tetrameric antibody complexes—a proprietary technology at Stem Cell. SpinSep uses centrifugation for immunodensity-based separation of cells. There is also an automated version of EasySep called RoboSep. R&D Systems' magnetic separation products for stem cells are the MagCellect CD105/Endoglin column and the MagCellect lineage depletion kits. The system enriches bone marrow samples for stem cells by removing lineage-committed cells such as T-cells and erythrocytes.
Stem cells are difficult to identify and isolate because they so closely resemble progenitor cells and differentiated adult cells. Future advances in knowledge about stem cells and the care and feeding of stem cells in vitro should yield great improvements in the ability to isolate and identify them for research and clinical purposes. In the meantime, high quality monoclonal antibodies and stem cell separation technologies offer realistic options for the laboratory.
References
1Balazs AB, et al. “Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow.” Blood, 107(6): 2317–2321, March 15, 2006.
