Appendix E: Stem Cell Markers

Appendix E.i. How Do Researchers Use Markers to Identify Stem Cells?

In recent years, scientists have discovered a wide array of stem cells that have unique capabilities to self-renew, grow indefinitely, and differentiate or develop into multiple types of cells and tissues. Researchers now know that many different types of stem cells exist but they all are found in very small populations in the human body, in some cases 1 stem cell in 100,000 cells in circulating blood. And, when scientists examine these cells under a microscope, they look just like any other cell in the tissue where they are found. So, how do scientists identify these rare type of cells found in many different cells and tissues—a process that is much akin to finding a needle in a haystack? The answer is rather simple thanks to stem cell "markers." This feature describes stem cell marker technology and how it is used in the research laboratory. Following this is a listing of some of the commonly used stem cell markers (see Appendix E.ii. Markers Commonly Used to Identify Stem Cells and to Characterize Differentiated Cell Types).

What are stem cell markers? Coating the surface of every cell in the body are specialized proteins, called receptors, that have the capability of selectively binding or adhering to other "signaling" molecules. There are many different types of receptors that differ in their structure and affinity for the signaling molecules. Normally, cells use these receptors and the molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body. These same cell surface receptors are the stem cell markers. Each cell type, for example a liver cell, has a certain combination of receptors on their surface that makes them distinguishable from other kinds of cells. Scientists have taken advantage of the biological uniqueness of stem cell receptors and chemical properties of certain compounds to tag or "mark" cells. Researchers owe much of the past success in finding and characterizing stem cells to the use of markers.

Stem cell markers are given short-hand names based on the molecules that bind to the stem cell surface receptors. For example, a cell that has the receptor stem cell antigen -1, on its surface, is identified as Sca-1. In many cases, a combination of multiple markers is used to identify a particular stem cell type. So now, researchers often identify stem cells in shorthand by a combination of marker names reflecting the presence (+) or absence (-) of them. For example, a special type of hematopoietic stem cell from blood and bone marrow called "side population" or "SP" is described as (CD34^-/low, c-Kit⁺, Sca-1⁺) [4].

Researchers use the signaling molecules that selectively adhere to the receptors on the surface of the cell as a tool that allows them to identify stem cells. Many years ago, a technique was developed to attach to the signaling molecule another molecule (or the tag) that has the ability to fluoresce or emit light energy when activated by an energy source such as an ultraviolet light or laser beam (see Figure E.i.1. Identifying Cell Surface Markers Using Fluorescent Tags). At the researchers' disposal are multiple fluorescent tags with emitted light that differ in color and intensity.

Figure E.i.1. Identifying Cell Surface Markers Using Fluorescent Tags.

(© 2001 Terese Winslow)

Described here are two approaches of how researchers use the combination of the chemical properties of fluorescence and unique receptor patterns on cell surfaces to identify specific populations of stem cells. One approach for using markers as a research tool is with a technique known as fluorescence-activated cell sorting (FACS) (see Figure E.i.2. Looking for a Needle in a Haystack: How Researchers Find Stem Cells) [1, 3, 5]. Researchers often use a FACS instrument to sort out the rare stem cells from the millions of other cells. With this technique, a suspension of tagged cells (i.e., bound to the cell surface markers are fluorescent tags) is sent under pressure through a very narrow nozzle—so narrow that cells must pass through one at a time. Upon exiting the nozzle, cells then pass, one-by-one, through a light source, usually a laser, and then through an electric field. The fluorescent cells become negatively charged, while nonfluorescent cells become positively charged. The charge difference allows stem cells to be separated from other cells. The researchers now have a population of cells that have all of the same marker characteristics, and with these cells they can conduct their research.

Figure E.i.2. Looking for a Needle in a Haystack: How Researchers Find Stem Cells.

(© 2001 Terese Winslow, Lydia Kibiuk, Caitlin Duckwall)

A second method uses stem cell markers and their fluorescent tags to visually assess cells as they exist in tissues. Often researchers want to assess how stem cells appear in tissues and in doing so they use a microscope to evaluate them rather than the FACS instrument. In this case, a thin slice of tissue is prepared, and the stem cell markers are tagged by the signaling molecule that has the fluorescent tag attached. The fluorescent tags are then activated either by special light energy or a chemical reaction. The stem cells will emit a fluorescent light that can easily be seen under the microscope.

Genetic and molecular biology techniques are extensively used to study how cells become specialized in the organism's development. In doing so, researchers have identified genes and transcription factors (proteins found within cells that regulate a gene's activity) that are unique in stem cells. Scientists use techniques such as polymerase chain reaction (PCR) to detect the presence of genes that are "active" and play a role guiding the specialization of a cell. This technique has is helpful to researchers to identify "genetic markers" that are characteristic of stem cells. For example, a gene marker called PDX-1 is specific for a transcription factor protein that initiates activation of the insulin gene. Researchers use this marker to identify cells that are able to develop islet cells in the pancreas.

Recently, researchers have applied a genetic engineering approach that uses fluorescence, but isn't dependent on cell surface markers. The importance of this new technique is that it allows the tracking of stem cells as they differentiate or become specialized. Scientists have inserted into a stem cell a "reporter gene" called green fluorescent protein or GFP [2]. The gene is only activated or "reports" when cells are undifferentiated and is turned off once they become specialized. Once activated, the gene directs the stem cells to produce a protein that fluoresces in a brilliant green color (see Figure E.i.3. Microscopic Image of Fluorescent-Labeled Stem Cell). Researchers are now coupling this reporting method with the FACS and microscopic methods described earlier to sort cells, identify them in tissues, and now, track them as they differentiate or become specialized.

Figure E.i.3. Microscopic Image of Fluorescent-Labeled Stem Cell.

(© 2001 Terese Winslow)

These discovery tools are commonly used in research laboratories and clinics today, and will likely play important roles in advancing stem cell research. There are limitations, however. One of them is that a single marker identifying pluripotent stem cells, those stem cells that can make any other cell, has yet to be found. As new types of stem cells are identified and research applications of them become increasingly complex, more sophisticated tools will be developed to meet investigators' needs. For the foreseeable future, markers will continue to play a major role in the rapidly evolving world of stem cell biology.

References

1. Bonner, W.A., Hulett, H.R., Sweet, R.G., and Herzenberg, L.A. (1972). Fluorescence activated cell sorting. Rev. Sci. Instrum. 43, 404–409.
2. Eiges, R., Schuldiner, M., Drukker, M., Yanuka, O., Itskovitz-Eldor, J., and Benvenisty, N. (2001). Establishment of human embryonic sten cell-transduced clones carrying a marker of undifferentiated cells. Curr. Biol. 11, 514–518.
3. Herzenberg, L.A. and De Rosa, S.C. (2000). Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine. Immunol. Today. 21, 383390.
4. Jackson, K., Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, and Goodell MA (2001). Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J. Clin. Invest. 107, 1–8.
5. Julius, M.H., Masuda, T., and Herzenberg, L.A. (1972). Demonstration that antigen-binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter. Proc. Natl. Acad. Sci. U. S. A. 69, 1934–1938.

 Appendix D | Table of Contents | Appendix F 

Historical content: June 17, 2001