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Initially proposed by Pappenheim in 1917 (1), the notion that stem cells are responsible for the renewal of the hematopoietic system was corroborated by several groups during the 1950s and 60s (2–5). This idea that adult tissue regeneration depends on a small population of resident cells has been more recently extended to other tissues such as epithelia, muscle, and the nervous system (6). Understanding the physiology of these cells has become one of the most exciting endeavors of biology today. Despite being conceptually simple, the definition of a stem cell is operationally challenging. A stem cell is defined as one that has the ability to selfrenew and the potential to give rise to several differentiated cell types; thus, to prove that a cell is indeed a stem cell, those two properties must be demonstrated. On the one hand, there is the need to observe the differentiation of the candidate cells; however, in doing so, they no longer are stem cells. On the other hand, there is the requirement that some of their progeny retain their original stem cell properties. Most of the experimental manipulations used to demonstrate potential to differentiate affect stem cells, changing their native properties. Furthermore, the exponential expansion of the differentiated progeny obscures the detection of the original stem cell population. Another hurdle that stem cell biologists have faced is the absence of a specific (i.e., present exclusively in stem cells) set of physical or chemical attributes. Several combinations of properties have been proposed and tested. However, at best, these allow the isolation of a subset of cells that is not truly a population of stem cells but a population that contains stem cells and that can be more or less enriched for stem cell activity. Needless to say, uncovering the exact phenotype of stem cells is one of the long-sought goals of stem cell biologists. The generally accepted model of stem cell physiology (Figure 1) considers the existence of a unique grandparent stem cell that gives rise to all the possible progeny, but there is little understanding about what happens in between. The model assumes that the abilities to divide and differentiate are the opposites of a spectrum; that is, the more differentiated a cell is, the less potential it has to divide. This is an attractive, simple, partially supported hypothesis, but there are exceptions to this rule, such as when fully differentiated cells divide to replace dead cells (e.g., in the liver). Another assumption this model makes is that there are cellular states between stem cells and fully differentiated cells that are variably called transit, transit amplifying, or progenitor cells and that every time a cell climbs down the differentiation tree, there is a fate decision involved. However, several matters remain unsettled. We do not know whether there is always a unique stem cell or several stem cells with (possibly) similar phenotype. If the latter is the case, then this could explain why populations of stem cells that are isolated using different strategies can display similar behavior, despite being phenotypically distinct. Furthermore, the properties of transit cells are also unknown. If commitment is reversible, then, as already mentioned, phenotypically distinct cells (because of differentiation) can end up having the same potential; if there are different types of transit cells, then some that behave as stem cells in one assay system may not perform well in another. Finally, the way commitment or fate is determined is still a mystery: is it a stochastic process, a consequence of intrinsic cell asymmetry, or an effect of the environment in which the cells dwell? In principle, given the right conditions (especially if the culture time is long enough), any developmental pathway could potentially be activated and give rise to any particular type of cell. Having all these issues in mind makes it possible to understand the limitations of most studies. In this work, we will review many of the techniques that are used to study adult stem cells, focusing on the hematopoietic stem cell (HSC) as a paradigm. We will begin by describing methods used to identify stem cells, including procedures used for their isolation, assays to study differentiation, and ways to assess self-renewal ability. The remainder of the article will deal with Review

Techniques for the Study of Adult Stem Cells: Be Fruitful and Multiply