Purification of hematopoietic stem cells for further biological study

For many years, the hematopoietic system has provided a convenient and fascinating model for studies of the molecular processes regulating cell growth and differentiation. However, this system also poses considerable challenges because the most primitive "stem" cells as well as their initial differentiating progeny are normally present in hematopoietic tissues at extremely low frequencies and no unique, stable phenotype has yet been identified to allow hematopoietic cells with specific stem and progenitor functions to be measured directly. Rather, this requires the use of functional assays that detect their developmental properties and take several weeks to complete. Accordingly, many investigations of primitive hematopoietic cell behavior and their responses to molecular cues in the environment have relied on the development of cell separation techniques specifically designed for obtaining highly enriched populations of primitive hematopoietic cells. Key to these procedures is the use of a preenrichment step(s) in which differences in cell density, size, or sensitivity to pharmacological agents or surface phenotype are exploited to first "debulk" the sample. This step can then be followed by a more selective antibody-mediated procedure to generate useful numbers of highly purified cells. Batchwise immunoadsorption techniques offer many advantages for obtaining enriched populations of hematopoietic progenitors because they avoid the nonspecific toxicity seen with antibody-mediated cell killing and are suitable for rapidly processing large samples. For any cell separation procedure, a balance must be struck between the purity and the recovery of the desired cells because steps to increase cell purity usually reduce yields. Both the negative and the positive selection techniques are useful strategies but negative selection usually requires one less manipulation step and circumvents potential effects incurred by the presence of antibody on the surface of the cell being isolated. Specific details for the use and results obtained with an immunomagnetic negative column selection technique are then presented.