**2. Development of methodology for HSC purification from mouse bone marrow**

In 1988, Spangrude et al tried to find a set of cell surface proteins that were associated with multi-lineage reconstitution ability, and succeeded to enrich such multipotential progenitors in the Lineage marker (Lin; generally including TER119, Mac1, Gr1, CD45R/B220, CD3, CD4, CD8)- Thy-1Low Sca-1+ fraction of mouse bone marrow (Spangrude et al, 1988). Indeed, they showed that only 30 Lin- Thy-1Low Sca-1+ cells injected via a tail vein could rescue 50% of lethally irradiated mice. Three years later, in 1991, Ogawa et al reported that hematopoietic progenitor activity of mouse bone marrow was excusive to the cells expressing c-kit, which is a receptor for stem cell factor (Ogawa et al., 1991). Since then, Lin- Sca-1+ c-kit+ (LSK) has been generally used as a canonical marker set for HSC enrichment.

It has been gradually recognized that the LSK fraction is heterogeneous, including longterm self-renewing HSC, short-term non-self-renewing HSC and lineage-committed progenitors. In 1996, Osawa et al reported that long-term HSC in adult bone marrow exist in the CD34 low to negative fraction among LSK cells (Osawa et al., 1996). Injection of a single CD34-/Low LSK cell resulted in multi-lineage long-term reconstitution in 21% of lethally irradiated mice whereas CD34+ LSK cells revealed early but only short-term reconstitution. Transplantation of graded numbers of CD34-/Low LSK cells showed that the CD34-/Low LSK fraction contains long-term HSC at the frequency of 1 out of 5 cells. In 2001, Christensen and Weissman also showed that the LSK fraction is heterogeneous and long-term HSC are highly enriched in the Flk2/Flt3 receptor tyrosine kinase negative cells (Christensen & Weissman, 2001).

In addition to the cell surface markers, another approach has been developed to enrich longterm HSC activity by focusing on their high efflux activity. Using the fluorescent DNAbinding dye Hoechst33342, in 1996, Goodell et al found that cells in a small Hoechstlowstained population (termed "Side population") can protect recipients from lethal irradiation at low cell doses (Goodell et al., 1996). A following study by Matsuzaki et al showed that, in combination with the CD34-/Low LSK phenotype, the strongest Hoechst33342 efflux activity (Tip-side population) can purify long-term multi-lineage HSC with almost absolute efficiency (Matsuzaki et al., 2004).

Recently, Morrison and colleagues reported an alternative method for HSC purification based on the expression pattern of the signaling lymphocytic activation molecule (SLAM) family proteins, i.e. CD150, CD244, and CD48 (Kiel et al., 2005). They showed that CD150+ CD48 cells were uniformly CD244 and a simple gating for CD150+ CD48- could enrich longterm HSC at approximately 1 in 5 cells. Moreover, combined with the canonical HSC marker LSK, the SLAM code could purify the HSC at 1 in 2 cells (Kiel et al., 2005).

Representative achievements during these 2 decades are summarized in Table 1. With surface markers, we can now purify the long-term multi-lineage HSC from adult mouse bone marrow with extremely high efficiency as Lin- Sca-1+ c-kit+ Thy1Low CD34-/low CD150+ CD48- cells. In fact, recent studies have demonstrated that the Lin- Sca-1+ c-kit+ CD34- CD150+ CD48- fraction in adult mouse bone marrow contains truly dormant HSC, which divide only 5-6 times during the life span (Wilson et al., 2008; Foudi et al., 2009).

In 1988, Spangrude et al tried to find a set of cell surface proteins that were associated with multi-lineage reconstitution ability, and succeeded to enrich such multipotential progenitors in the Lineage marker (Lin; generally including TER119, Mac1, Gr1, CD45R/B220, CD3, CD4, CD8)- Thy-1Low Sca-1+ fraction of mouse bone marrow (Spangrude et al, 1988). Indeed, they showed that only 30 Lin- Thy-1Low Sca-1+ cells injected via a tail vein could rescue 50% of lethally irradiated mice. Three years later, in 1991, Ogawa et al reported that hematopoietic progenitor activity of mouse bone marrow was excusive to the cells expressing c-kit, which is a receptor for stem cell factor (Ogawa et al., 1991). Since then, Lin- Sca-1+ c-kit+ (LSK) has been generally used as a canonical

It has been gradually recognized that the LSK fraction is heterogeneous, including longterm self-renewing HSC, short-term non-self-renewing HSC and lineage-committed progenitors. In 1996, Osawa et al reported that long-term HSC in adult bone marrow exist in the CD34 low to negative fraction among LSK cells (Osawa et al., 1996). Injection of a single CD34-/Low LSK cell resulted in multi-lineage long-term reconstitution in 21% of lethally irradiated mice whereas CD34+ LSK cells revealed early but only short-term reconstitution. Transplantation of graded numbers of CD34-/Low LSK cells showed that the CD34-/Low LSK fraction contains long-term HSC at the frequency of 1 out of 5 cells. In 2001, Christensen and Weissman also showed that the LSK fraction is heterogeneous and long-term HSC are highly enriched in the Flk2/Flt3 receptor tyrosine kinase negative cells

In addition to the cell surface markers, another approach has been developed to enrich longterm HSC activity by focusing on their high efflux activity. Using the fluorescent DNAbinding dye Hoechst33342, in 1996, Goodell et al found that cells in a small Hoechstlowstained population (termed "Side population") can protect recipients from lethal irradiation at low cell doses (Goodell et al., 1996). A following study by Matsuzaki et al showed that, in combination with the CD34-/Low LSK phenotype, the strongest Hoechst33342 efflux activity (Tip-side population) can purify long-term multi-lineage HSC with almost absolute

Recently, Morrison and colleagues reported an alternative method for HSC purification based on the expression pattern of the signaling lymphocytic activation molecule (SLAM) family proteins, i.e. CD150, CD244, and CD48 (Kiel et al., 2005). They showed that CD150+

term HSC at approximately 1 in 5 cells. Moreover, combined with the canonical HSC marker

Representative achievements during these 2 decades are summarized in Table 1. With surface markers, we can now purify the long-term multi-lineage HSC from adult mouse bone marrow with extremely high efficiency as Lin- Sca-1+ c-kit+ Thy1Low CD34-/low CD150+

CD150+ CD48- fraction in adult mouse bone marrow contains truly dormant HSC, which

LSK, the SLAM code could purify the HSC at 1 in 2 cells (Kiel et al., 2005).

CD48- cells. In fact, recent studies have demonstrated that the Lin-

divide only 5-6 times during the life span (Wilson et al., 2008; Foudi et al., 2009).

and a simple gating for CD150+ CD48-

could enrich long-

Sca-1+ c-kit+ CD34-

**2. Development of methodology for HSC purification from mouse bone** 

**marrow** 

marker set for HSC enrichment.

(Christensen & Weissman, 2001).

efficiency (Matsuzaki et al., 2004).

cells were uniformly CD244-

CD48-


\*Tip-SP: The highest Hoechst-efflux fraction in the Side Population

Table 1. Markers for hematopoietic stem cells in adult mouse bone marrow
