The procedure for the purification of HSCs has made great progress along with the identification of molecular markers that characterize the cells having reconstitution activities in transplanted mice. The most primitive murine HSCs are considered to be with the CD34low-c-Kit+ Sca-1+Lin- (CD34-KSL) phenotype, since a single cell with this phenotype could reconstitute whole hematopoiesis in vivo with high probability . In addition to the specific surface phenotype, HSCs present in steady-state adult mouse BM are functionally characterized by their ability to excrete Rhodamine-123 and Hoechst 33342 [6, 7]. When adult mouse BM cells are stained with Hoechst 33342, exposed to the UV light, and examined at 2 emission wavelengths simultaneously, HSCs are found in the rare side population (SP) with the dim fluorescence because of this ability [8, 9]. The low fluorescence of HSCs after staining with Rhodamine-123 and Hoechst 33342 is attributed to their selective expression of different ABC transporters, P-glycoprotein and bcrp-1, respectively [10,11].
In addition, the cells having the strongest dye efflux capacity (Tip-SP cells) with the CD34-KSL phenotype were shown to be the most primitive HSCs, which can reconstitute long-term hematopoiesis with almost 100% probability even after the single cell transplantation . The cells in the SP fraction is considered to be in G0 phase, and this state is supposed to be regulated by "hematopoietic niche'' in the BM as described later. On the other hand, in steady-state human BM, a majority of HSCs having long-term reconstitution activity express CD34 , and the most primitive human HSCs are considered to exist in the population with Lineage (CD3, CD4, CD8, CD11b, CD19, CD20, hCD56, and glycophorin A)-CD34+CD38- phenotype . Whereas CD34+ has been utilized as a marker of HSCs, recent reports indicated that SRCs (SCID-repopulating cells) are more concentrated in CD133+ cells than in CD34+ cells [15,16]. That is, Suzuki et al. demonstrated that CB CD133-sorted cells contained an approximately 4.5-fold greater absolute number of SRCs than CD34-sorted cells . Further clinical studies comparing CD34+ and CD133+ cells would determine which is a better phenotype to collect and evaluate human HSCs.
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