PU.1 is expressed in functional HSCs with long-term reconstituting potential. (A) Sorting gates for the SP fraction. The vast majority of Lin^–Sca-1⁺c-Kit⁺CD34^– cells possessed the SP profile. (B) RT-PCR analysis of PU.1 mRNA in CD34⁺ or CD34^– SP HSCs. HPRT indicates hypoxanthin phosphoribosyltransferase. (C) Expression of GFP in HSCs and GMPs purified from PU.1^GFP/+ mice. The vast majority of Lin^–Sca-1⁺c-Kit⁺ HSCs and SP HSCs expressed PU.1-GFP irrespective of CD34 expression at the single-cell level. GMPs expressed GFP at a higher level compared with HSCs. (D) Ly5.2⁺ donor type Gr-1⁺, CD3⁺, and B220⁺ cells were successfully reconstituted in a mouse that received a transplant of a single CD34^–PU.1-GFP⁺ HSC. Multilineage reconstitution has been maintained for more than 20 weeks.