Quantitative proteomics can be used in a discovery-based manner to identify new potential stem cell-surface protein markers. In order to validate the usefulness of any potential cell-surface markers (whether protein, lipid, carbohydrate, etc.), the candidate markers will then need to enter into an iterative cycle of validation and discovery (), which includes techniques other than proteomics. An essential question is how specific is the cell-surface protein marker, and separately, is the marker restricted to stem/progenitor cells or does it show lineage restrictions. To address these questions, an antibody that reacts with the antigen will have to be generated or obtained. Thus, it is essential that the proteomic efforts for discovering the candidate protein markers provide as much characterization as possible about the protein, so that epitope selection is efficient. This will aid in the development of antibodies with sufficient specificity, which is typically labor and time intensive. Secondarily and if not already established, it is essential to determine if the marker is quantitatively more or less abundant on original stem/progenitor cells relative to other cells. A variety of techniques is available for this, but the use of genomic and proteomic databases are a good place to begin. Once this has been established, it is critical to determine if the marker(s) is present on a subpopulation of cells present in vivo, particularly if the starting population of stem/progenitor cells involved in vitro cultivation. In vitro cultivation can lead to changes (epigenetic) or even abnormalities (transformations) in the cell’s properties. An in vivo correlate must therefore be found using immunostaining and/or flow cytometry. If the latter is successful, then fluorescence-activated cell sorting (FACS) or analogous techniques can be used to isolate antigen-presenting cells. Once the cells are isolated and characterized by a variety of potential in vitro assays (colony forming, proliferation, differentiation), it will then be possible to determine if the isolated cells can self-renew and differentiate (i.e. are really stem cells). Finally, the cells should be tested for therapeutic functionality in animal models. Primary isolates (but ideally with a marker gene) need to be introduced into an appropriate animal models (injury models and testing for repair, transgenic models with gene deletions) to ensure that they function like stem cells (i.e. participate in chimera formation, integrate into appropriate niche, or differentiate into the appropriate progeny). The ability of the isolated cells to self-renew and produced known (and unknown) progeny in vivo can then be verified, thus setting the stage for eventual therapeutic trials.
Figure 4 Roadmap to identifying and validating stem cell surface markers. Iterative process of discovery and validation required to define a panel of positive and negative selection markers useful in defining stem cells and purifying homogeneous cell populations. (more ...)
It is through this rigorous process that stem cell-surface protein markers identified in the discovery phase become bona fide markers for future research or clinical applications. Ultimately, a panel of antibodies will be necessary to isolate cells to homogeneity that have a specific differentiation/developmental potential. It should then be possible to isolate resident stem or progenitor cells that are neither tumorogenic nor immunologically incompatible with a host. Once achieved, then specific types of stem cells my be available to improve motion or appendage movement in spinal cord injuries, improve function and ejection fraction in heart failure or after injury (myocardial infarction), or improve insulin responsiveness and normalization of glucose levels in diabetes. It is likely that the process from initial discovery to final validation will involve numerous iterations to arrive at a suitable panel of negative and positive selection markers and their respective antibodies. While this process is perhaps rather time consuming, it currently holds the best hope for identifying and isolating primary stem/progenitor cells capable of treating intractable diseases without inadvertent complications.