The vertebrate hematopoietic system produces at least eight distinct lineages of mature blood cells in a continuous manner throughout adult life. These lineages include red blood cells, monocytic, granulocytic and basophilic myeloid cells, the B and T-cell compartments of the antigen-specific immune system, as well as the megakaryocytes of the thrombopoietic system. Continuous replenishment of the mature cell populations is necessitated by their finite half-lives. In order to meet daily demands, the rate of mature blood cell production is very high; in the human, many billions of new cells are produced every day. There are a number of good reviews which cover the basic features of hematopoietic system biology (Metcalf et al. 1997, Domen & Weissman 1999). At the heart of the hematopoietic system is a rare population of undifferentiated stem cells which are collectively responsible for life-long blood cell production. The hematopoietic stem cell is characterized by two cardinal properties. First, they are multipotential; that is, individual stem cells can clonally produce all of the different cell types found in the blood (Dexter 1987, Morrison 1995). Second, at least operationally, they are self-renewing; that is, during the proliferation of stem cells commitment/differentiation is balanced by the production of additional stem cells (Potten & Loeffler 1990, Jordan & Lemischka 1991). This self-renewal ability follows logically from the lifelong production of mature cell populations. In the murine system it has been shown that single stem cells are both necessary and sufficient for the establishment of a normally functioning hematopoietic system (Smith et al 1992, Jordan & Lemischka 1991, Osawa et al. 1996). Throughout adult life, the stem cell population resides in the bone marrow. It is thought that the precursors of these adult stem cells originate either in the extraembryonic yolk sac or, more likely, in the embryo proper (Zon 1995, Dzierzak et al. 1995). The first tissue where definitive, fully functional stem cell populations are detectable in reasonable numbers is the midgestation fetal liver (Moore et al. 1997, Harrison & Astle 1997). In this tissue, the stem cell population expands and shortly before birth, the fetal liver hematopoietic stem cells migrate to the bone marrow and take-up residence there. An additional property of stem cells is their rare absolute frequency in either adult or fetal tissue sources. It has been estimated that only one in 10⁴ to 10⁵ bone marrow or fetal liver cells is a stem cell (Harrison & Astle 1997). Therefore, an adult mouse which contains a number on the order of 10⁸ total bone marrow cells has a complement of 1,000 to 10,000 stem cells. Similar stem cell frequencies are observed in the fetal liver. Although many invitro assay systems have been proposed to measure the stem cell population, the only definitive way to reveal the existence and the properties of the most primitive stem cell population is by in vivo transplantation. In general, this involves the transfer of a functional hematopoietic system into recipient animals whose endogenous hematopoietic system has been ablated by radiation. The definition of a stem cell includes the ability to function over very long (greater than or equal to six months) post transplantation time frames. Other recipient systems have also been utilized; most notably, genetically anemic and stem cell deficient mutant mice which lack the ckit receptor tyrosine kinase (Abramson et al. 1977, Trevisan et al. 1996, Migliaccio et al. 1999). Regardless of the exact experimental design, most in vivo stem cell assay systems yield very consistent results. This provides a high degree of confidence that the fundamental properties of the stem cell are governed, at least to a significant degree, by mechanisms which are cell-intrinsic. Specifically, the properties of multipotentiality and self-renewal are revealed and not defined by the specific assay system. Importantly, the biology of stem cells is also governed by microenvironmental mechanisms (Moore et al. 1997, Aiuti et al. 1998). Extensive details describing the properties of hematopoietic stem cells can be found in several good recent reviews (Fuchs & Segre 2000, Weissman 2000, Morrison et al. 1997). The overall organization of the hematopoietic system is hierarchical. As the most primitive stem cell begins to divide, it produces more committed progenitor cell entities (Suda et al. 1984). In many cases, these progenitor cells are still multipotential in their differentiation capacities, but have a significantly reduced self-renewal ability (Osawa et al. 1996). Such cells can be measured in a variety of in vitro culture systems and also by their ability to function in vivo over short time intervals (Osawa et al. 1996). An additional feature of the hematopoietic stem/progenitor hierarchy is that progenitor cell numbers are directly correlated with their degree of commitment. That is, the most primitive stem cells are the least abundant while the most committed progenitors are the most abundant. Consistent with this, in the bone marrow the most primitive stem cells are likely to be quiescent or cycling at a very slow rate (Fleming et al. 1993, Bradford et al. 1997, Cheshier et al. 1999). In contrast, a much higher proportion of fetal liver stem cells is in active cell cycle (Fleming et al. 1993, Ema and Nakauchi 2000).      (back to top)

In all cases, stem cell assays are indirect and retroactive. Simply stated, the existence and the properties of a stem cell population are inferred solely by the analysis of their mature cell progeny. While this can be accomplished with great precision using clonal markers such as karyotype differences or recombinant retroviruses, it is still a major limitation (Lemischka 1992).

A major advance in the stem cell field came with the development of strategies to physically purify transplantable activity from bone marrow and from fetal liver (Spangrude et al. 1988, Jordan et al. 1990). In essence, an elusive biological activity was endowed with a physical phenotype, and therefore, became a cellular entity. In general, stem cell purification relies on the judicious combination of cell surface markers none of which are by themselves specifically expressed by the stem cell. All of these markers are defined by monoclonal antibody reagents previously derived using other cell types. These reagents can serve as either positive or negative markers for stem cell activity. Details of various purification procedures can be found in a number of good primary articles and reviews (Spangrude & Johnson 1990, Morrison et al. 1995, Osawa et al. 1996, Ziegler et al. 1999, Trevisan & Iscove 1995). Other stem cell purification strategies take advantage of differential staining with vital dyes such Rhodamine-123 or Hoechst 33342. Low-intensity staining (due to low uptake in the case of Hoechst and rapid efflux in the case of Rh-123) of these dyes are properties associated with bone marrow stem cells (Wolf et al. 1993, Leemhuis et al. 1996, Goodell et al. 1996).

A general issue with all stem cell purification strategies is that while transplantable activity can be greatly enriched (usually about 1,000 to 2,000-fold), the purified cell populations are still heterogeneous. In most cases, it is difficult to separate the most primitive stem cell compartment from closely related primitive progenitors. Recently, some efforts have suggested that this will ultimately be possible (Jones et al. 1990, Jones et al. 1996, Osawa et al. 1996, Morrison et al. 1997, Kondo et al. 1997, Akashi et al. 2000 ). Other considerations, most notably the need to rely on a complex in vivo assay system, suggest that it may not be possible to rigorously address the exact degree of stem cell homogeneity in any physically purified cell population.

The hematopoietic system is not unique in its ability to produce large populations of mature cells throughout life. The skin epithelium, the intestinal epithelium and the male germ line also share this property (Fuchs & Segre 2000, Weissman 2000, Morrison et al. 1997). Other tissues such as the liver, the muscle, the vasculature, and possibly, the central and peripheral nervous systems have the ability to replenish mature cell populations in response to injury or stress (Thorgeirsson 1996, Overturf et al. 1997, Schultz et al. 1994, Gage et al. 1995, McKay 1997, Johansson et al. 1999, Doetsch et al. 1999, Tropepe et al. 2000, Morrison et al. 1999). In some cases, candidate stem cells from these tissues have been identified and analyzed, although not as extensively as in the hematopoietic system (Li et al. 1998, Watt 1998, Potten 1998, Bach et al. 2000, Morrison et al. 1999). Until recently, the prevailing dogma held that these stem cells identified from different tissue sources would be "dedicated" to the specific tissue. That is, a hematopoietic stem cell would only be capable of blood production, the muscle stem cell would only be capable of differentiating into muscle, etc. Very recently, this dogma has been challenged in several reports (for commentary see Lemischka 1999). Highly enriched hematopoietic stem cell populations have been shown to possess muscle differentiation ability and muscle cells demonstrate the capability of reconstituting the entire complement of blood cell lineages (Gussoni et al. 1999, Jackson et al. 1999). Most remarkably, stem cells from the central nervous system can also function to generate a blood cell system upon transplantation into irradiated animals (Bjornson et al. 1999). These observations suggest that the developmental flexibility of stem cell populations isolated from different sources may be greater than previously thought. An obvious extension of this notion is that the genetic programs of stem cells isolated from different tissue sources may be general, and thus, more responsible for the maintenance of a developmentally plastic state than for directing a particular set of differentiation fates.

SCDb Scheme