Phenotypic analysis of G-CSF– or G-CSF plus SCF–mobilized PB primitive CD34⁺(CD34⁺CD38⁻Lin⁻) or CD34⁻(CD34⁻CD38⁻AC133⁺Lin⁻) cells after 10 days in in vitro culture.

G-CSF–mobilized (A) or G-CSF plus SCF–mobilized (B) PB Lin⁻ cells were sorted by means of 3-color flow cytometry with the use of antibodies for human CD34, CD38, and AC133. Lin⁻ cells were sorted for CD34 expression (Ai,Bi), with sorting gates established to select for CD34⁻CD38⁻ (R1) and CD34⁺CD38⁻ (R2) populations. Cells were re-sorted to exclude any potential contaminating cells, and each population was reanalyzed for AC133 expression. CD34⁻CD38⁻AC133⁺Lin⁻(Aii,Bii) and CD34⁺CD38⁻Lin⁻(Aiii, Biii) cells were purified for in vitro culture. Equal numbers of purified CD34⁻CD38⁻AC133⁺Lin⁻and CD34⁺CD38⁻Lin⁻ cells were cultured in serum-free conditions containing hematopoietic growth factors. After 10 days of culture, the contents of the wells were analyzed for CD34, CD38, and AC133 expression. The CD34 and CD38 expression is shown for 10-day–cultured CD34⁺CD38⁻Lin⁻ (Aiv,Biv) and CD34⁻CD38⁻AC133⁺Lin⁻(Av,Bv) cells for G-CSF–mobilized (A) and G-CSF plus SCF–mobilized (B) PB samples. Insets illustrate isotype (IgG1) controls of stained cultured cells used to establish quadrant statistics for CD34⁻CD38⁻ (R3) and CD34⁺CD38⁻cells (R4) gating. Approximately 3.3% ± 1.1% of the G-CSF plus SCF–mobilized CD34⁻CD38⁻AC133⁺Lin⁻cells acquired CD34 expression after 10 days of culture, while G-CSF–mobilized CD34⁻CD38⁻AC133⁺Lin⁻cells did not (0.1%). The AC133 expression after 10 days in vitro culture is shown for CD34⁻CD38⁻AC133⁺Lin⁻(Avi,Bvi) and CD34⁺CD38⁻Lin⁻(Avi,Bvi) cells. Data shown are representative of independent experiments with the use of a G-CSF sample and the mean of 3 additional G-CSF plus SCF–mobilized PB samples.